Final Report Summary - MARS (Marker Assisted Resistance to Sharka)
Executive Summary:
Sharka, caused by the Plum Pox Virus (PPV), is the most devastating disease affecting stone fruit trees in Europe. In order to respond to this critical situation, the FP7-funded SharCo project (2008-2012) devoted a significant part of its efforts on the development of first-generation PPV resistant plant materials, guidelines for new plantings and molecular tools for the implementation of marker assisted selection (MAS) in apricot breeding programs. The first steps of establishment of MAS in SharCo presented some limitations that hampered the EU-wide implementation of a high throughput MAS approach:
1. While most of the PPV resistant individuals displayed the resistant alleles, a significant number of PPV susceptible individuals showed the same resistant haplotype. We thus hypothesize that the presence of other factors or genes involved in the mechanism of resistance to sharka in apricot could explain these unexpected results.
2. Screening of apricot progenies for both PPV resistance and local adaptation to different fruit tree cultivations was limited to few hundreds of individuals due to technical and financial issues.
In this context, the current proposal, MARS (for Marker Assisted Resistance to Sharka), was designed to boost the production of sharka resistant stone fruit cultivars by improving and transferring efficient and reliable procedures of MAS to SMEs.
First, molecular tools developed in SharCo were upgraded for multiplexing before transfer to SMEs. They then served in the screening of over thirty thousands of apricot progenies in which sharka resistance was combined with locally adapted, high value varieties. About half of the progenies showed resistant alleles over the first locus linked to PPV resistance in SharCo. In the meantime, part of the MARS consortium focused on 1) the identification of the second genetic factor controlling PPV resistance, 2) the development of a new set of molecular markers linked to this second locus and 3) the validation of these new tools before transfer to SMEs. A new set of four molecular markers, complementary to the ones issued from SharCo, were developed. After validation on a subset of apricot progenies and multiplexing, they were also transferred to SMEs. They were then used to screen the promising apricot progenies selected beforehands with the SharCo’s tools. They also served to screen progenies in which at least two sources of resistance were combined. In parallel to those molecular approaches, part of the consortium worked on pyramiding several sources of resistance in new breeding programs as well as evaluating the agronomical added-value of the first true resistant material identified both in SharCo and MARS. Moreover, dissemination activities were conducted, targeting fruit producers, nurserymen and extension services, in order to promote the plantation and cultivation of PPV resistant apricot cultivars developed by the MARS partners.
By the end of the MARS project, we can claim on an efficient translation of MAS to SMEs and the implementation of high throughput selection of PPV resistant apricot cultivars adapted for cultivation all over Europe. Such an approach is contributing significantly to building efficient and durable resistance to sharka disease at the European level.
Project Context and Objectives:
- MARS context and rationale
Sharka, caused by the Plum Pox Virus (PPV), is the most devastating disease affecting fruit trees in Europe (cost of 10 billion Euros in the last 30 years for disease management) and has put the world fruit tree industry in a precarious position. In order for Europe to provide breeders with the tools needed to respond to this critical situation, the EC funded the “SharCo” project (small Collaborative FP7 project) from 2008-2012. SharCo developed tools such as first-generation PPV resistant plant materials, accurate and reliable methods of PPV detection, guidelines for new plantings, an early warning systems and a decision-support system. Moreover, SharCo devoted part of its effort to the integrated study of genetic resistance to PPV, thus developing molecular tools and knowledge for implementation of marker assisted selection in apricot breeding programs. Molecular marker assisted selection, often simply referred to as marker assisted selection (MAS), involves selection, through molecular markers, of plants carrying genomic regions that are involved in the expression of traits of interest. Whereas at SharCo project end in 2012, part of the genetic tools and plant material were ready for transfer to European breeding programs and private stakeholders such as SMEs, however there were limitations on the translation of these results to realized economic value. This was due to: (i) the restricted number of resistance donors used in SharCo limiting the number of resistance genes available for pyramiding, (ii) the lack of a high throughput method for screening thousands of promising breeding materials which combine both resistance to sharka and locally adapted, high quality value traits. Indeed, although natural sources of resistance have been identified and used in apricots in SharCo and other European breeding programs, they are too limited in number to secure the development of durable sharka resistance, EU-wide. In SharCo, we also demonstrated that some of the parents used for European breeding programmes harbour a restricted number of genetic factors linked to PPV resistance and thus they are only partially resistant to the disease. In the long term this poses a problem of for durable resistance in newly released apricot cultivars.
In this context, the overall aim of MARS was to boost the production of sharka resistant stone fruit cultivars by transferring the efficient and reliable procedures of MAS conceived in SharCo to SMEs to enable targeted, high throughput selection of PPV resistant apricot cultivars adapted for cultivation all over Europe.
- MARS scientific and technical objectives
One of the simplest approaches to controlling the spread and impact of PPV is the development of fruit tree varieties with significant levels of sustainable resistance to the pathogen. For this purpose, the MARS consortium devoted a significant part of its efforts to the integrated study of genetic resistance to PPV, thus developing molecular tools and knowledge for implementation of marker assisted selection (MAS) in apricot breeding programs. However, while developing pathogen resistant new varieties, breeders need also to satisfy consumers taste and meet growers and industry constraints for the production of fruits with optimized flavor, color, texture and resistance to damage during transport. Therefore, it is essential to select PPV resistant material together with these quality criteria in varieties that simultaneously show good productivity and good local adaptation. To maintain their economic position among temperate tree fruits producing countries in the world, stone fruit breeders need to rapidly introduce a high level of resistance to sharka, into high quality value varieties, locally adapted to their environment and cultivation conditions. This process is significantly hampered by the complexities inherent to tree breeding. The first important step is the identification of adequate parents and the development of molecular tools allowing fast and efficient screening of progenies for PPV resistance or for other traits of interest. Plant material issued from controlled crosses between PPV-resistant parents and locally adapted, agronomically valuable varieties is the ultimate of such breeding programmes in order to meet consumers and fruit producers requirements.
Overall, the strategic objective of MARS was to transfer to European SMEs the novel methods and tools originating from SharCo, in order to enable the acceleration of the selection of sharka resistant plant material. For that purpose, MARS aimed at:
- Producing and upgrading reliable molecular tools for MAS in view of improving resistance of plants propagated in nurseries and cultivated in orchards by:
o Identifying molecular markers linked to a maximum of genetic factors involved in PPV resistance;
o Reinforcing plant resistance by combining multiple genetic factors in ’elite’ germplasm and pre-competitive breeding material;
- establishing a high throughput protocol for MAS transferable to, and applicable by, various end-users such as SMEs/laboratories specialized in biotechnology, diagnosis, legal certification of new cultivars as well as breeding companies of the public and private sectors;
- demonstrating the MAS technology within SMEs established in Europe and Turkey to streamline and accelerate the introgression of resistance into new varieties by:
o Validating the molecular tools in thousands of progenies set up in various European environmental conditions of cultivation and viral inoculums pressure.
MARS also relied on the SMEs and their respective local network of producers, extension services and growers unions to disseminate the first released apricot varieties and promote the multiplication and planting of sharka-resistant material following the cultivation guidelines established by SharCo.
Project Results:
1) From conventional breeding to marker assisted selection of PPV resistant material
- Towards the improvement of tools for Marker Assisted Selection
Resistance to PPV in apricot is controlled by few genetic factors and most of the trait variance is explained by one major QTL (Quantitative Trait Locus, corresponding to the major genomic region linked to the resistance trait) named PPVres. This major QTL is mapping on the upper arm of linkage group one (LG1) and explains by itself up to 70% of the resistance trait variance. A set of three markers (PGS1.21 ZP002 and PGS1.24) targeting the PPVres locus was developed in the frame of SharCo (Soriano et al. 2012; Decroocq et al. 2014). In the latest study, we demonstrated that this first marker set developed SharCo is not broadly applicable for Marker Assisted Selection (MAS) and that marker assisted breeding based on the sole PPVres locus is not sufficient to unambiguously select PPV resistant apricot cultivars. Whereas the major locus is essential for the outcome of viral infection leading to resistance or susceptibility, complete and stable expression of the trait requires at least a second locus (Decroocq et al. 2014). In fact, while most of the PPV resistant individuals displayed the resistant alleles, a significant number of PPV susceptible individuals showed the same resistant haplotype. We thus hypothesize that the presence of other r genes involved in the mechanism of resistance to sharka in apricot could explain these unexpected results. Implementing MAS of PPV resistance in apricot will thus require the identification and targeting with molecular markers of the other locus/loci that potentially modulate the effect of PPVres. We thus focused on identifying markers linked to the other genetic factor(s) and to use this data to optimize MAS for resistance to sharka.
For this purpose, we implemented two complementary strategies: 1) a Genome Wide Association Study (GWAS) and 2) a QTL mapping approach in 5 distinct F1 segregating populations. Mapping of the other genetic factors controlling resistance to sharka is described in details in the milestone MS1 (available on the MARS collaborative platform).
1. Genome-wide association studies on cultivated and wild accessions
Two distinct loci were identified (Figure 1), which are partly in LD (linkage disequilibrium) and are both mapping on LG1 (linkage group 1 or chromosome 1) (Mariette et al. 2015): One around 10-11 Mbp (could correspond to MetaPPV1b) (Marandel et al, 2009) and one at 8 Mbp (possibly corresponding to PPVres) (Soriano et al, 2012), the most significant being the first one and surprisingly, not PPVres.
Because of the physical proximity of both loci and of the significant LD between them, few recombinants are expected in segregating populations which might not help to distinguish the effect of each locus, their epistatic effect (if any). Thus, ideally, the development of markers should not be in LD, to allow targeting both loci independently, which is not the case in cultivated apricot germplasm. In consequence, a new, complementary approach, based on GWAS in wild, natural populations of apricot segregating for resistance to PPV was initiated. However, due to the time needed for phenotyping, results will be available only by the end of the project.
• 2. Screening of populations from controlled crosses between cultivated apricot accessions and new QTL analyses
Both at INRA-Avignon (partner P01b) and UNIBO (partner P03), segragating populations which have been already phenotyped were screened for selected markers in the sharka resistance region (upper part of chromosome 1), among which markers developed in the above study. The analysis showed a smaller, although significant peak centred on the position 31 Mbp or 6 Mbp which might be the target for future investigation (Figure 2). After adding the new SNP and SSR markers in the above apricot map frameworks, QTL analysis was performed once again. However, results did not change significantly, compared to the previous ones; no additional region was identified despite the higher marker coverage on linkage group 1 and, at a lower extent, linkage groups 3 and 4. In our opinion, these results are likely due to the genomic structure of the populations (F1), a relatively low number of individuals that does not allow for breaking linkage and discriminating close QTL regions, if any, and the linkage disequilibrium between the two QTL, PPVres and metaPPV1b.
At this stage, the next step was then to develop new molecular tools based on the above data and targeting the other factor(s) of resistance, beside PPVres. A set of 68 SNP and 42 SSR markers were developed during the first period of MARS (see list of markers and details in Deliverable D1.1). However, when developing markers for MAB in heterozygous plant species, the following criteria are essential: i) high polymorphism of the markers selected, ii) co-dominance of the markers allowing the scoring of both alleles of a diploid, heterozygous genome, iii) good transferability from one breeding population to another. Following those rules, we made the choice to develop SSR (Single Sequence Repeat, also called microsatellite) markers as we did in SharCo for the first set of markers. A set of four SSR markers was selected (see Deliverable D1.1 and milestone MS2, available on the MARS collaborative platform), based on data obtained on the mapping populations in France and in Italy (see Figure 3 for a representation of the second set of markers, relative to the one developed in SharCo).
Figure 3: Schematic diagram of the upper part of chromosome 1 in apricot showing the positions of the different molecular markers.
In red are represented the markers that belong to the first set of markers developed in SharCo (PPVres locus); in black, markers implemented in MARS. Positions of the SSR and SSLP markers are depicted over the figure, in base pairs (bp) from the chromosome start. They correspond to the relative position of the forward primer in the peach annotated genome sequence v1.0 (www.rosacea.org). Chr 1: Chromosome 1.
Conditions of multiplexing were set up in collaboration with the Biotech SMEs, before being used in routine in the second period of the project. While transferring this second set of markers to SMEs, we also tried to validate them in a subset of progenies for which we already knew their resistance or susceptibility status. Indeed, the new set of molecular markers was tested in apricot populations previously phenotyped in France, Italy and Spain (Table 1). However, those studies showed that while the second set of molecular markers helped in some cases to sort out true susceptible progenies that were initially mislabelled as resistant, it did not solve all problems described in Decroocq et al. 2014. Indeed, it appears that additional work will be needed for the development of molecular markers able to screen efficiently seedlings derived from crossing in which alternative sources of resistance such as ‘Harcot’ are used as parent.
Table 1: Screening of extensive populations of apricot issued from one single source of resistance, with the two sets of molecular markers implemented in MARS
- To build up a durable resistance to PPV
In the last two years of SharCo, various donors of resistance were challenged with eight PPV isolates, representing five viral strains. Few accessions (‘SEO’, ‘Lito’, ‘Harlayne’ and ‘Stella’) remained resistant whatever the strain was. This result confirms that in this material, more than one genetic factor participate to the trait. Therefore, MARS also focused on: 1) pyramiding several sources of resistance in breeding populations, thus combining the different factors of resistance in few genotypes, and 2) applying the two sets of molecular markers (one coming from SharCo and the second one developed in the course of MARS see above) on this new material.
Two types of activities were conducted for this purpose in MARS: 1) the characterization of progenies issued from crosses between two sources of resistance and obtained previously to the start of the MARS project (some of them, in the course of SharCo), 2) the crossing between at least two sources of resistance by some MARS partners. The new crosses were performed in the first year of the project (2014). A total of 12,020 seedlings in which at least two sources of resistance were cumulated (see complete list in Deliverable D1.2 and updated in WP01’s second periodic report) were genotyped with the two sets of markers (Table 2). Part of them were also tested for resistance to sharka in greenhouses in order to verify their level of resistance in comparison with seedlings issued from one single source of resistance.
Table 2: Seedlings scored so far for resistance gene pyramiding and results obtained for the two sets of markers implemented in MARS.
A given seedling was scored as genotypically ‘resistant’ when at least one resistant allele is detected (heterozygous status) at each tested locus.
All together those preliminary data show that the frequency of resistant siblings is significantly higher to the ones issued from one single source of resistance (66.98% for 43% on average for PPVres). Full details on this task and results obtained by the MARS consortium are presented in deliverable D1.2. Evaluation of the durability and stability of this complex resistance in the obtained breeding material is still ongoing and will require several vegetative cycles.
- Diversification of the sources of resistance to sharka
Most of the sources of resistance used up to now as parents (donors of the resistance) in European conventional breeding programs share the same genetic origin, a Central-Asian material introduced in North-American breeding programs over 50 years ago. Therefore, regardless to the implementation of high throughput MAB, we cannot rule out that identified resistances may be overcome, in the future, by new virus strains. Therefore, it was important to diversify those sources of resistance and identify new, still-unknown germplasm for future breeding programmes. The above two sets of molecular markers were used to survey the apricot germplasm as well as local, old/ancient cultivars, to try to identify additional resistant parents or varieties.
Two types of plant material were gathered for this task (see complete list into the Deliverable D1.3):
1) Cultivated, current or ancient landraces kept at the French National genetic resource collection, located at INRA, Avignon (174 accessions) and at UMIL (P02) (132 accessions);
2) Apricot accessions from wild, natural populations together with local ancient cultivars and landraces originating from Central Asia and the Irano-Caucasian region (552 accessions currently maintained by partner P01, INRA-Bdx, UMR BFP). The sampling of the second type of plant material was performed in the last 5 years in the frame of the STONE FP7 IRSES project (coordinated by partner P01a).
A summary of the results is presented in the Tables 3 and 4 below as well as in the deliverable D1.3 together with a full list of individuals screened. It showed a higher proportion of PPVres resistant alleles in the apricot natural populations and in landraces originating from the area of origin and domestication of this species.
Table 3: Marker Assisted screening of the irano-caucasian and central Asian apricot germplasm
Table 4: Marker Assisted screening of modern and old/ancient apricot cultivated germplasm
A subset of this original material was tested for resistance to sharka by infecting them with PPV-Marcus strain (Figure 3 for the wild germplasm).
Figure 3: Distribution of the first 39 wild apricot accessions according to their mean viral accumulation value as a percentage of optical density value, over two vegetative cycles. Individuals are ordered from the lowest to the highest. Accessions are considered as resistant when their mean OD value is inferior or equal to the mean OD value of the negative control (green dotted line). The red box displays the most probable, highly resistant wild accessions.
In summary, in the apricot collections of MARS partners, there are, up to now, 858 apricot accessions available. 153 out of the 858 are bearing alleles associated to PPV resistance according to the three PPVres markers. However, after phenotypic validation, in total, we confirmed 25 new sources of resistance (8 in the wild germplasm and 16 in the National collections of apricot cultivars). The new putative parents will be made available to European breeders in order to improve durability of PPV resistance in the future cultivars by combining different sources of resistance.
2) Establishment and transfer to SMEs of a high throughput marker assisted technology of selection of PPV resistant apricot trees
Europe is characterized by its large variability in agronomical and environmental conditions of stone fruit cultivation, from continental climates characterized by cold winter periods to Mediterranean, arid areas with mild winter periods. This means that, when setting up the production of PPV resistant material for re-planting, it is essential to cover the diversity of agro-ecological conditions of apricot cultivation in Europe. Because of this diversity in environmental conditions, we need to optimize the selection of sharka resistant material which has to be adapted to various European agronomical conditions. In consequence, in MARS, we set up the screening of thousands of apricot progenies, in order to be in adequation with various local cultivation conditions and market requirements, which obviously vary from Mediterranean regions to Eastern European countries. This explains the establishment in MARS of a high-throughput protocol of MAS, for the screening of several thousands of apricot breeding material and the selection of new cultivars adapted to the diversity of apricot production in Europe.
Besides building an efficient and durable resistance to sharka disease at the European level, the MARS project also aimed at boosting the production of sharka resistant stone fruit cultivars by transferring the above procedures of MAS to SMEs to enable targeted, high throughput selection of PPV resistant apricot cultivars adapted for cultivation all over Europe. First of all, a standardised protocol was established to ease the running of molecular analyzes in different biotech laboratories located in different environments, on several thousands of apricot seedlings (Deliverable D2.1). This protocol was implemented in laboratories in France, Greece and Turkey, initially with the first set of markers developed in SharCo and then for the second set, developed in MARS. In total, 31,584 apricot seedlings were screened initially with the first set of markers (Deliverable D2.2). Material that was scored as resistant in deliverable D2.2 was then tested with the second set of markers, in order to validate and refine the previous data (Deliverable D2.3). Results of this high throughput screening for PPV resistant apricot seedlings are depicted in Table 5, below. Overall, only 9.35% of the seedlings displayed resistant alleles for the two sets of markers, at the seven marker loci, for 54.96% when cumulating at least two sources of resistance (see Table 2).
Table 5: PPV resistant apricot progenies scored with the first and second set of markers
This promising material was or is currently being transferred to nurseries for agronomical evaluation.
3) Selection of new PPV resistant cultivars adapted to local conditions of apricot cultivation and with a high quality and commercial value
In order to fit the fruit market requirement as well as various cultivation conditions, in parallel to the translation of the tools and methodology to SMEs, a portion of the MARS effort was directed at understanding how to develop a durable resistance to PPV in any European and extra-European orchard. All along the procedure, we kept on feeding the MAS programs established by the SMEs in order to optimize the selection of sharka resistant material adapted to various European agronomical conditions and consumers’ taste. For this purpose, we established procedures of agronomical evaluation of putatively resistant apricot pre-breeding material. Agronomical trait evaluation covered from the floral biology, to the fructification, the fruit quality, the yield as well as the tree features (vigour, growth habit, thining and tree management…). Many of those tasks related to the agronomical evaluation of promising, PPV resistant apricot seedlings are still ongoing but orchards were established in each partner country (Deliverable D2.4). A total of 15 varieties resistant to sharka were released in the course of MARS as described below in Table 6.
Table 6: PPV resistant apricot accessions currently under development in the MARS nurseries
In summary, MARS delivered the following key results within the 2 years of the project work-plan (in brackets, corresponding deliverable):
1. Validated tools for SMEs:
• Set of molecular markers developed in SharCo and validated in MARS (D1.1). This tool was produced by targeting the main locus controlling the resistance trait in apricot donors;
• Optimized set of molecular markers targeting the other genetic factor linked to the resistance (D1.1). This new set will complement the one developed in SharCo in order to implement a more reliable and broad-spectrum technology of MAS;
2. apricot parents and hybrids which display a high level of resistance to PPV (D1.2 and D1.3);
3. recommendations for breeders:
• for the establishment of MAS for obtaining PPV resistant varieties (D2.1);
• for the crosses of PPV resistant parents with locally adapted cultivars (D2.2);
4. Standards for the planting of PPV resistant apricot varieties EU-wide, other than in Turkey (D2.3).
Potential Impact:
- Expected impact of the implementation of a highthrough-put Marker Assisted Selection for resistance to sharka in apricot
Attracting end-users such as breeding companies for the selection of PPV resistant fruit trees
The development of Marker Assisted Selection is expected to attract end-users such as private or public breeding companies, European- and world-wide. Up to now, to test an interesting cultivar, one needs 4 years of monitoring after infection to assess the level of resistance. This slows the breeding process, as it is a labour-intensive, time consuming and expensive process the selection of material resistant to PPV . In woody plants, molecular tools have the potential to give us early information on the genetics of Prunus progenies and marker-assisted selection is the only short-term solution to rapidly select individuals resistant to sharka disease.
The production and maintenance of a minimum of 6,000 hybrids in an apricot breeding programme costs about 30,000 Euros per year (data provided by nursery SME CEP Innovation). Knowing that the evaluation of PPV resistant material requires a minimum of 4 years, switching from traditional breeding to MAS would allow saving at least 120,000 Euros, per breeding programme. For 8 countries involved in apricot breeding programmes in Europe, MAS would allow saving about 1 million Euro for each year all over Europe.
By the end of the MARS project, a new set of molecular markers and guidelines for the implementation of MAS for resistance to sharka will be proposed to public and private breeders and certification services (legal offices of registration of new cultivars from the CPVO – European Community Plant Variety Office), it will promote the use and plantation of PPV resistant varieties.
Sustainibility of the stone fruit industry
Plantation of PPV resistant cultivars EU-wide is expected to help not only in the management of the sharka disease but also in the sustainable production of apricot fruits and industries (mostly dried fruits in Mediterannean and Eastern rural areas. Transfer of the resistance mechanism(s) available in apricot to other stone fruit species such as diploid plum could also be facilitated, in the long term, with the help of molecular markers linked to the apricot genetic determinants. Indeed, MAS is useful for backcross breeding for the introgression of resistance genes from wild or distantly related species while selecting against the undesirable characteristics of the wild or distinct species. Backcross of the resistance to diploid plum and then to peach is thus ‘feasible’, at least if molecular markers are available and through the implementation of high throughput MAS. However, it will still require years and several generations to achieve it.
In order to accelerate the selection of sharka resistant and agronomically valuable fruit trees for cultivar deployment, all this plant material and molecular tools was combined to switch from conventional breeding to marker assisted breeding. MARS thus brought methodology, innovative molecular tools and breeding expertise together for the deployment of resistance in apricot cultivars circulating in Europe or under selection.
- Promotion of PPV resistant breeding and pre-breeding material for plantation
One whole workpackage (Dissemination, or WP3) aimed at putting at the disposal of breeders, nurserymen and fruit producers, resistant apricot plant material limiting the impact of the Sharka disease. For this purpose, part of the MARS activities were devoted to the promotion of sharka resistant breeding material to a range of key stakeholders, including decision makers from industry, early-stage & experienced researchers, farmers, policy makers and consumers. Know-how and plant material will be used to draw standards for the deployment of PPV resistant material. MARS’ results was also disseminated through the MARS dedicated website (www.inra.fr/mars) as well as the establishment of a PPV resistance genotyping database (http://users.unimi.it/apricotmap).
We thus aimed at bridging this gap by organising training and research workshops on PPV with special attention paid to end-users such as fruit producers and nurserymen, in Western and European countries. Leaflets, in nationale language, were produced and distributed to gardening shops and nurseries as well as leaflets describing agronomical, morphological and sensorial quality features of newly produced or/and ready-to-be released, sharka resistant cultivars (see Deliverable . They were transferred to farmers, nurseries and extension services by MARS partners; and uploaded on the MARS website, for further dissemination and printing.
Four types of dissemination activities were performed in the course of MARS, as follows:
• Training workshops on the multiplication and cultivation of PPV resistant apricot material were organised during the first period of MARS, in Spain and Romania, in national languages. Other workshops were organised over the second period, in Spain, Bulgaria, Turkey, Greece, Italy and France. They addressed to fruit producers, managers of major nurseries and advisers from Agricultural Extension Services all over Europe (see all details, copies of the work programme and minutes of the day in the WP3’s second periodic report, available on the MARS collaborative platform). For a complete list of training workshops that took place in the second period, see Table 7 or Deliverable D3.2.
Table 7: MARS training workshops organised by the MARS partners
The MARS leaflet produced in the first period by partners P12 and P06 was translated in national languages and disseminated during their respective training workshops or within their stakeholders’ networks.
• Research Institutions and SMEs participated at meetings and orchard visits under Plant Protection Service and their own organizations, respectively. They were mainly dedicated to PPV control strategies, their effectiveness, their cost-effectiveness, and the end-users perception, including the development of resistant cultivars.
• MARS last dissemination event was organised concomitant to the Fruit Attraction fair, at IFEMA, Madrid, on the 29th of October 2015. Three communications were delivered:
- Welcome and Introduction of the MARS project by Véronique Decroocq (INRA, partner P01)
- The Marker Assisted Selection technology: Legacy of the SharCo FP7 project by Daniele Bassi and Filippo Geuna (UMIL, partner P02)
- The MARS results by Fabienne Moreau (ADNid, partner P10) and Manuel Rubio (CEBAS-CSIC, partner P08)
MARS partners also presented the outcomes of the MARS project in their own breeding or research program, through the display of posters, on site. All communications and posters are available online at www.inra.fr/mars/.
• Publications in peer-reviewed international journals as well as local, vulgarisation newspapers and participation to international symposium and conferences, especially to the World Expo in Milano, where the outcomes of the FP7 SharCo and MARS projects were presented at the EU pavilion, on the 15th of July 2015 (see From SharCo to MARS: A translational research path to solve a major phytosanitary problem http://europa.eu/expo2015/node/731). See the complete list of dissemination tools in the section Use and Dissemination of the Foreground.
- A portal and a database for Marker Assisted Selection of PPV resistant apricot trees
A relational database, based on the 4D (4th Dimension) software has been designed in order to gather and store MARS genotyping data. This database was set up for running under the Windows 2012 Server operating system on an HP Proliant server hosted at the University of Milano (partner P02, UMIL) (see Deliverable D3.3). The database has been fed with genotyping data from the first set of markers developed in SharCo. The process of feeding up the database with data from the second set of markers is still ongoing. Those data are coming from around 25,090 individuals which after screening with the two sets of molecular markers provided 71,608 genotypes. The database is accessible and queryable by the partners and, ultimately (once initial results are suitably published), will be opened to the international community, once the last batch of data is uploaded. An hyperlink will be placed on the international Genome Database for Rosaceae (GDR) (www.rosaceae.org).
List of Websites:
www.inra.fr/mars
Coordinator: decroocq@bordeaux.inra.fr
Sharka, caused by the Plum Pox Virus (PPV), is the most devastating disease affecting stone fruit trees in Europe. In order to respond to this critical situation, the FP7-funded SharCo project (2008-2012) devoted a significant part of its efforts on the development of first-generation PPV resistant plant materials, guidelines for new plantings and molecular tools for the implementation of marker assisted selection (MAS) in apricot breeding programs. The first steps of establishment of MAS in SharCo presented some limitations that hampered the EU-wide implementation of a high throughput MAS approach:
1. While most of the PPV resistant individuals displayed the resistant alleles, a significant number of PPV susceptible individuals showed the same resistant haplotype. We thus hypothesize that the presence of other factors or genes involved in the mechanism of resistance to sharka in apricot could explain these unexpected results.
2. Screening of apricot progenies for both PPV resistance and local adaptation to different fruit tree cultivations was limited to few hundreds of individuals due to technical and financial issues.
In this context, the current proposal, MARS (for Marker Assisted Resistance to Sharka), was designed to boost the production of sharka resistant stone fruit cultivars by improving and transferring efficient and reliable procedures of MAS to SMEs.
First, molecular tools developed in SharCo were upgraded for multiplexing before transfer to SMEs. They then served in the screening of over thirty thousands of apricot progenies in which sharka resistance was combined with locally adapted, high value varieties. About half of the progenies showed resistant alleles over the first locus linked to PPV resistance in SharCo. In the meantime, part of the MARS consortium focused on 1) the identification of the second genetic factor controlling PPV resistance, 2) the development of a new set of molecular markers linked to this second locus and 3) the validation of these new tools before transfer to SMEs. A new set of four molecular markers, complementary to the ones issued from SharCo, were developed. After validation on a subset of apricot progenies and multiplexing, they were also transferred to SMEs. They were then used to screen the promising apricot progenies selected beforehands with the SharCo’s tools. They also served to screen progenies in which at least two sources of resistance were combined. In parallel to those molecular approaches, part of the consortium worked on pyramiding several sources of resistance in new breeding programs as well as evaluating the agronomical added-value of the first true resistant material identified both in SharCo and MARS. Moreover, dissemination activities were conducted, targeting fruit producers, nurserymen and extension services, in order to promote the plantation and cultivation of PPV resistant apricot cultivars developed by the MARS partners.
By the end of the MARS project, we can claim on an efficient translation of MAS to SMEs and the implementation of high throughput selection of PPV resistant apricot cultivars adapted for cultivation all over Europe. Such an approach is contributing significantly to building efficient and durable resistance to sharka disease at the European level.
Project Context and Objectives:
- MARS context and rationale
Sharka, caused by the Plum Pox Virus (PPV), is the most devastating disease affecting fruit trees in Europe (cost of 10 billion Euros in the last 30 years for disease management) and has put the world fruit tree industry in a precarious position. In order for Europe to provide breeders with the tools needed to respond to this critical situation, the EC funded the “SharCo” project (small Collaborative FP7 project) from 2008-2012. SharCo developed tools such as first-generation PPV resistant plant materials, accurate and reliable methods of PPV detection, guidelines for new plantings, an early warning systems and a decision-support system. Moreover, SharCo devoted part of its effort to the integrated study of genetic resistance to PPV, thus developing molecular tools and knowledge for implementation of marker assisted selection in apricot breeding programs. Molecular marker assisted selection, often simply referred to as marker assisted selection (MAS), involves selection, through molecular markers, of plants carrying genomic regions that are involved in the expression of traits of interest. Whereas at SharCo project end in 2012, part of the genetic tools and plant material were ready for transfer to European breeding programs and private stakeholders such as SMEs, however there were limitations on the translation of these results to realized economic value. This was due to: (i) the restricted number of resistance donors used in SharCo limiting the number of resistance genes available for pyramiding, (ii) the lack of a high throughput method for screening thousands of promising breeding materials which combine both resistance to sharka and locally adapted, high quality value traits. Indeed, although natural sources of resistance have been identified and used in apricots in SharCo and other European breeding programs, they are too limited in number to secure the development of durable sharka resistance, EU-wide. In SharCo, we also demonstrated that some of the parents used for European breeding programmes harbour a restricted number of genetic factors linked to PPV resistance and thus they are only partially resistant to the disease. In the long term this poses a problem of for durable resistance in newly released apricot cultivars.
In this context, the overall aim of MARS was to boost the production of sharka resistant stone fruit cultivars by transferring the efficient and reliable procedures of MAS conceived in SharCo to SMEs to enable targeted, high throughput selection of PPV resistant apricot cultivars adapted for cultivation all over Europe.
- MARS scientific and technical objectives
One of the simplest approaches to controlling the spread and impact of PPV is the development of fruit tree varieties with significant levels of sustainable resistance to the pathogen. For this purpose, the MARS consortium devoted a significant part of its efforts to the integrated study of genetic resistance to PPV, thus developing molecular tools and knowledge for implementation of marker assisted selection (MAS) in apricot breeding programs. However, while developing pathogen resistant new varieties, breeders need also to satisfy consumers taste and meet growers and industry constraints for the production of fruits with optimized flavor, color, texture and resistance to damage during transport. Therefore, it is essential to select PPV resistant material together with these quality criteria in varieties that simultaneously show good productivity and good local adaptation. To maintain their economic position among temperate tree fruits producing countries in the world, stone fruit breeders need to rapidly introduce a high level of resistance to sharka, into high quality value varieties, locally adapted to their environment and cultivation conditions. This process is significantly hampered by the complexities inherent to tree breeding. The first important step is the identification of adequate parents and the development of molecular tools allowing fast and efficient screening of progenies for PPV resistance or for other traits of interest. Plant material issued from controlled crosses between PPV-resistant parents and locally adapted, agronomically valuable varieties is the ultimate of such breeding programmes in order to meet consumers and fruit producers requirements.
Overall, the strategic objective of MARS was to transfer to European SMEs the novel methods and tools originating from SharCo, in order to enable the acceleration of the selection of sharka resistant plant material. For that purpose, MARS aimed at:
- Producing and upgrading reliable molecular tools for MAS in view of improving resistance of plants propagated in nurseries and cultivated in orchards by:
o Identifying molecular markers linked to a maximum of genetic factors involved in PPV resistance;
o Reinforcing plant resistance by combining multiple genetic factors in ’elite’ germplasm and pre-competitive breeding material;
- establishing a high throughput protocol for MAS transferable to, and applicable by, various end-users such as SMEs/laboratories specialized in biotechnology, diagnosis, legal certification of new cultivars as well as breeding companies of the public and private sectors;
- demonstrating the MAS technology within SMEs established in Europe and Turkey to streamline and accelerate the introgression of resistance into new varieties by:
o Validating the molecular tools in thousands of progenies set up in various European environmental conditions of cultivation and viral inoculums pressure.
MARS also relied on the SMEs and their respective local network of producers, extension services and growers unions to disseminate the first released apricot varieties and promote the multiplication and planting of sharka-resistant material following the cultivation guidelines established by SharCo.
Project Results:
1) From conventional breeding to marker assisted selection of PPV resistant material
- Towards the improvement of tools for Marker Assisted Selection
Resistance to PPV in apricot is controlled by few genetic factors and most of the trait variance is explained by one major QTL (Quantitative Trait Locus, corresponding to the major genomic region linked to the resistance trait) named PPVres. This major QTL is mapping on the upper arm of linkage group one (LG1) and explains by itself up to 70% of the resistance trait variance. A set of three markers (PGS1.21 ZP002 and PGS1.24) targeting the PPVres locus was developed in the frame of SharCo (Soriano et al. 2012; Decroocq et al. 2014). In the latest study, we demonstrated that this first marker set developed SharCo is not broadly applicable for Marker Assisted Selection (MAS) and that marker assisted breeding based on the sole PPVres locus is not sufficient to unambiguously select PPV resistant apricot cultivars. Whereas the major locus is essential for the outcome of viral infection leading to resistance or susceptibility, complete and stable expression of the trait requires at least a second locus (Decroocq et al. 2014). In fact, while most of the PPV resistant individuals displayed the resistant alleles, a significant number of PPV susceptible individuals showed the same resistant haplotype. We thus hypothesize that the presence of other r genes involved in the mechanism of resistance to sharka in apricot could explain these unexpected results. Implementing MAS of PPV resistance in apricot will thus require the identification and targeting with molecular markers of the other locus/loci that potentially modulate the effect of PPVres. We thus focused on identifying markers linked to the other genetic factor(s) and to use this data to optimize MAS for resistance to sharka.
For this purpose, we implemented two complementary strategies: 1) a Genome Wide Association Study (GWAS) and 2) a QTL mapping approach in 5 distinct F1 segregating populations. Mapping of the other genetic factors controlling resistance to sharka is described in details in the milestone MS1 (available on the MARS collaborative platform).
1. Genome-wide association studies on cultivated and wild accessions
Two distinct loci were identified (Figure 1), which are partly in LD (linkage disequilibrium) and are both mapping on LG1 (linkage group 1 or chromosome 1) (Mariette et al. 2015): One around 10-11 Mbp (could correspond to MetaPPV1b) (Marandel et al, 2009) and one at 8 Mbp (possibly corresponding to PPVres) (Soriano et al, 2012), the most significant being the first one and surprisingly, not PPVres.
Because of the physical proximity of both loci and of the significant LD between them, few recombinants are expected in segregating populations which might not help to distinguish the effect of each locus, their epistatic effect (if any). Thus, ideally, the development of markers should not be in LD, to allow targeting both loci independently, which is not the case in cultivated apricot germplasm. In consequence, a new, complementary approach, based on GWAS in wild, natural populations of apricot segregating for resistance to PPV was initiated. However, due to the time needed for phenotyping, results will be available only by the end of the project.
• 2. Screening of populations from controlled crosses between cultivated apricot accessions and new QTL analyses
Both at INRA-Avignon (partner P01b) and UNIBO (partner P03), segragating populations which have been already phenotyped were screened for selected markers in the sharka resistance region (upper part of chromosome 1), among which markers developed in the above study. The analysis showed a smaller, although significant peak centred on the position 31 Mbp or 6 Mbp which might be the target for future investigation (Figure 2). After adding the new SNP and SSR markers in the above apricot map frameworks, QTL analysis was performed once again. However, results did not change significantly, compared to the previous ones; no additional region was identified despite the higher marker coverage on linkage group 1 and, at a lower extent, linkage groups 3 and 4. In our opinion, these results are likely due to the genomic structure of the populations (F1), a relatively low number of individuals that does not allow for breaking linkage and discriminating close QTL regions, if any, and the linkage disequilibrium between the two QTL, PPVres and metaPPV1b.
At this stage, the next step was then to develop new molecular tools based on the above data and targeting the other factor(s) of resistance, beside PPVres. A set of 68 SNP and 42 SSR markers were developed during the first period of MARS (see list of markers and details in Deliverable D1.1). However, when developing markers for MAB in heterozygous plant species, the following criteria are essential: i) high polymorphism of the markers selected, ii) co-dominance of the markers allowing the scoring of both alleles of a diploid, heterozygous genome, iii) good transferability from one breeding population to another. Following those rules, we made the choice to develop SSR (Single Sequence Repeat, also called microsatellite) markers as we did in SharCo for the first set of markers. A set of four SSR markers was selected (see Deliverable D1.1 and milestone MS2, available on the MARS collaborative platform), based on data obtained on the mapping populations in France and in Italy (see Figure 3 for a representation of the second set of markers, relative to the one developed in SharCo).
Figure 3: Schematic diagram of the upper part of chromosome 1 in apricot showing the positions of the different molecular markers.
In red are represented the markers that belong to the first set of markers developed in SharCo (PPVres locus); in black, markers implemented in MARS. Positions of the SSR and SSLP markers are depicted over the figure, in base pairs (bp) from the chromosome start. They correspond to the relative position of the forward primer in the peach annotated genome sequence v1.0 (www.rosacea.org). Chr 1: Chromosome 1.
Conditions of multiplexing were set up in collaboration with the Biotech SMEs, before being used in routine in the second period of the project. While transferring this second set of markers to SMEs, we also tried to validate them in a subset of progenies for which we already knew their resistance or susceptibility status. Indeed, the new set of molecular markers was tested in apricot populations previously phenotyped in France, Italy and Spain (Table 1). However, those studies showed that while the second set of molecular markers helped in some cases to sort out true susceptible progenies that were initially mislabelled as resistant, it did not solve all problems described in Decroocq et al. 2014. Indeed, it appears that additional work will be needed for the development of molecular markers able to screen efficiently seedlings derived from crossing in which alternative sources of resistance such as ‘Harcot’ are used as parent.
Table 1: Screening of extensive populations of apricot issued from one single source of resistance, with the two sets of molecular markers implemented in MARS
- To build up a durable resistance to PPV
In the last two years of SharCo, various donors of resistance were challenged with eight PPV isolates, representing five viral strains. Few accessions (‘SEO’, ‘Lito’, ‘Harlayne’ and ‘Stella’) remained resistant whatever the strain was. This result confirms that in this material, more than one genetic factor participate to the trait. Therefore, MARS also focused on: 1) pyramiding several sources of resistance in breeding populations, thus combining the different factors of resistance in few genotypes, and 2) applying the two sets of molecular markers (one coming from SharCo and the second one developed in the course of MARS see above) on this new material.
Two types of activities were conducted for this purpose in MARS: 1) the characterization of progenies issued from crosses between two sources of resistance and obtained previously to the start of the MARS project (some of them, in the course of SharCo), 2) the crossing between at least two sources of resistance by some MARS partners. The new crosses were performed in the first year of the project (2014). A total of 12,020 seedlings in which at least two sources of resistance were cumulated (see complete list in Deliverable D1.2 and updated in WP01’s second periodic report) were genotyped with the two sets of markers (Table 2). Part of them were also tested for resistance to sharka in greenhouses in order to verify their level of resistance in comparison with seedlings issued from one single source of resistance.
Table 2: Seedlings scored so far for resistance gene pyramiding and results obtained for the two sets of markers implemented in MARS.
A given seedling was scored as genotypically ‘resistant’ when at least one resistant allele is detected (heterozygous status) at each tested locus.
All together those preliminary data show that the frequency of resistant siblings is significantly higher to the ones issued from one single source of resistance (66.98% for 43% on average for PPVres). Full details on this task and results obtained by the MARS consortium are presented in deliverable D1.2. Evaluation of the durability and stability of this complex resistance in the obtained breeding material is still ongoing and will require several vegetative cycles.
- Diversification of the sources of resistance to sharka
Most of the sources of resistance used up to now as parents (donors of the resistance) in European conventional breeding programs share the same genetic origin, a Central-Asian material introduced in North-American breeding programs over 50 years ago. Therefore, regardless to the implementation of high throughput MAB, we cannot rule out that identified resistances may be overcome, in the future, by new virus strains. Therefore, it was important to diversify those sources of resistance and identify new, still-unknown germplasm for future breeding programmes. The above two sets of molecular markers were used to survey the apricot germplasm as well as local, old/ancient cultivars, to try to identify additional resistant parents or varieties.
Two types of plant material were gathered for this task (see complete list into the Deliverable D1.3):
1) Cultivated, current or ancient landraces kept at the French National genetic resource collection, located at INRA, Avignon (174 accessions) and at UMIL (P02) (132 accessions);
2) Apricot accessions from wild, natural populations together with local ancient cultivars and landraces originating from Central Asia and the Irano-Caucasian region (552 accessions currently maintained by partner P01, INRA-Bdx, UMR BFP). The sampling of the second type of plant material was performed in the last 5 years in the frame of the STONE FP7 IRSES project (coordinated by partner P01a).
A summary of the results is presented in the Tables 3 and 4 below as well as in the deliverable D1.3 together with a full list of individuals screened. It showed a higher proportion of PPVres resistant alleles in the apricot natural populations and in landraces originating from the area of origin and domestication of this species.
Table 3: Marker Assisted screening of the irano-caucasian and central Asian apricot germplasm
Table 4: Marker Assisted screening of modern and old/ancient apricot cultivated germplasm
A subset of this original material was tested for resistance to sharka by infecting them with PPV-Marcus strain (Figure 3 for the wild germplasm).
Figure 3: Distribution of the first 39 wild apricot accessions according to their mean viral accumulation value as a percentage of optical density value, over two vegetative cycles. Individuals are ordered from the lowest to the highest. Accessions are considered as resistant when their mean OD value is inferior or equal to the mean OD value of the negative control (green dotted line). The red box displays the most probable, highly resistant wild accessions.
In summary, in the apricot collections of MARS partners, there are, up to now, 858 apricot accessions available. 153 out of the 858 are bearing alleles associated to PPV resistance according to the three PPVres markers. However, after phenotypic validation, in total, we confirmed 25 new sources of resistance (8 in the wild germplasm and 16 in the National collections of apricot cultivars). The new putative parents will be made available to European breeders in order to improve durability of PPV resistance in the future cultivars by combining different sources of resistance.
2) Establishment and transfer to SMEs of a high throughput marker assisted technology of selection of PPV resistant apricot trees
Europe is characterized by its large variability in agronomical and environmental conditions of stone fruit cultivation, from continental climates characterized by cold winter periods to Mediterranean, arid areas with mild winter periods. This means that, when setting up the production of PPV resistant material for re-planting, it is essential to cover the diversity of agro-ecological conditions of apricot cultivation in Europe. Because of this diversity in environmental conditions, we need to optimize the selection of sharka resistant material which has to be adapted to various European agronomical conditions. In consequence, in MARS, we set up the screening of thousands of apricot progenies, in order to be in adequation with various local cultivation conditions and market requirements, which obviously vary from Mediterranean regions to Eastern European countries. This explains the establishment in MARS of a high-throughput protocol of MAS, for the screening of several thousands of apricot breeding material and the selection of new cultivars adapted to the diversity of apricot production in Europe.
Besides building an efficient and durable resistance to sharka disease at the European level, the MARS project also aimed at boosting the production of sharka resistant stone fruit cultivars by transferring the above procedures of MAS to SMEs to enable targeted, high throughput selection of PPV resistant apricot cultivars adapted for cultivation all over Europe. First of all, a standardised protocol was established to ease the running of molecular analyzes in different biotech laboratories located in different environments, on several thousands of apricot seedlings (Deliverable D2.1). This protocol was implemented in laboratories in France, Greece and Turkey, initially with the first set of markers developed in SharCo and then for the second set, developed in MARS. In total, 31,584 apricot seedlings were screened initially with the first set of markers (Deliverable D2.2). Material that was scored as resistant in deliverable D2.2 was then tested with the second set of markers, in order to validate and refine the previous data (Deliverable D2.3). Results of this high throughput screening for PPV resistant apricot seedlings are depicted in Table 5, below. Overall, only 9.35% of the seedlings displayed resistant alleles for the two sets of markers, at the seven marker loci, for 54.96% when cumulating at least two sources of resistance (see Table 2).
Table 5: PPV resistant apricot progenies scored with the first and second set of markers
This promising material was or is currently being transferred to nurseries for agronomical evaluation.
3) Selection of new PPV resistant cultivars adapted to local conditions of apricot cultivation and with a high quality and commercial value
In order to fit the fruit market requirement as well as various cultivation conditions, in parallel to the translation of the tools and methodology to SMEs, a portion of the MARS effort was directed at understanding how to develop a durable resistance to PPV in any European and extra-European orchard. All along the procedure, we kept on feeding the MAS programs established by the SMEs in order to optimize the selection of sharka resistant material adapted to various European agronomical conditions and consumers’ taste. For this purpose, we established procedures of agronomical evaluation of putatively resistant apricot pre-breeding material. Agronomical trait evaluation covered from the floral biology, to the fructification, the fruit quality, the yield as well as the tree features (vigour, growth habit, thining and tree management…). Many of those tasks related to the agronomical evaluation of promising, PPV resistant apricot seedlings are still ongoing but orchards were established in each partner country (Deliverable D2.4). A total of 15 varieties resistant to sharka were released in the course of MARS as described below in Table 6.
Table 6: PPV resistant apricot accessions currently under development in the MARS nurseries
In summary, MARS delivered the following key results within the 2 years of the project work-plan (in brackets, corresponding deliverable):
1. Validated tools for SMEs:
• Set of molecular markers developed in SharCo and validated in MARS (D1.1). This tool was produced by targeting the main locus controlling the resistance trait in apricot donors;
• Optimized set of molecular markers targeting the other genetic factor linked to the resistance (D1.1). This new set will complement the one developed in SharCo in order to implement a more reliable and broad-spectrum technology of MAS;
2. apricot parents and hybrids which display a high level of resistance to PPV (D1.2 and D1.3);
3. recommendations for breeders:
• for the establishment of MAS for obtaining PPV resistant varieties (D2.1);
• for the crosses of PPV resistant parents with locally adapted cultivars (D2.2);
4. Standards for the planting of PPV resistant apricot varieties EU-wide, other than in Turkey (D2.3).
Potential Impact:
- Expected impact of the implementation of a highthrough-put Marker Assisted Selection for resistance to sharka in apricot
Attracting end-users such as breeding companies for the selection of PPV resistant fruit trees
The development of Marker Assisted Selection is expected to attract end-users such as private or public breeding companies, European- and world-wide. Up to now, to test an interesting cultivar, one needs 4 years of monitoring after infection to assess the level of resistance. This slows the breeding process, as it is a labour-intensive, time consuming and expensive process the selection of material resistant to PPV . In woody plants, molecular tools have the potential to give us early information on the genetics of Prunus progenies and marker-assisted selection is the only short-term solution to rapidly select individuals resistant to sharka disease.
The production and maintenance of a minimum of 6,000 hybrids in an apricot breeding programme costs about 30,000 Euros per year (data provided by nursery SME CEP Innovation). Knowing that the evaluation of PPV resistant material requires a minimum of 4 years, switching from traditional breeding to MAS would allow saving at least 120,000 Euros, per breeding programme. For 8 countries involved in apricot breeding programmes in Europe, MAS would allow saving about 1 million Euro for each year all over Europe.
By the end of the MARS project, a new set of molecular markers and guidelines for the implementation of MAS for resistance to sharka will be proposed to public and private breeders and certification services (legal offices of registration of new cultivars from the CPVO – European Community Plant Variety Office), it will promote the use and plantation of PPV resistant varieties.
Sustainibility of the stone fruit industry
Plantation of PPV resistant cultivars EU-wide is expected to help not only in the management of the sharka disease but also in the sustainable production of apricot fruits and industries (mostly dried fruits in Mediterannean and Eastern rural areas. Transfer of the resistance mechanism(s) available in apricot to other stone fruit species such as diploid plum could also be facilitated, in the long term, with the help of molecular markers linked to the apricot genetic determinants. Indeed, MAS is useful for backcross breeding for the introgression of resistance genes from wild or distantly related species while selecting against the undesirable characteristics of the wild or distinct species. Backcross of the resistance to diploid plum and then to peach is thus ‘feasible’, at least if molecular markers are available and through the implementation of high throughput MAS. However, it will still require years and several generations to achieve it.
In order to accelerate the selection of sharka resistant and agronomically valuable fruit trees for cultivar deployment, all this plant material and molecular tools was combined to switch from conventional breeding to marker assisted breeding. MARS thus brought methodology, innovative molecular tools and breeding expertise together for the deployment of resistance in apricot cultivars circulating in Europe or under selection.
- Promotion of PPV resistant breeding and pre-breeding material for plantation
One whole workpackage (Dissemination, or WP3) aimed at putting at the disposal of breeders, nurserymen and fruit producers, resistant apricot plant material limiting the impact of the Sharka disease. For this purpose, part of the MARS activities were devoted to the promotion of sharka resistant breeding material to a range of key stakeholders, including decision makers from industry, early-stage & experienced researchers, farmers, policy makers and consumers. Know-how and plant material will be used to draw standards for the deployment of PPV resistant material. MARS’ results was also disseminated through the MARS dedicated website (www.inra.fr/mars) as well as the establishment of a PPV resistance genotyping database (http://users.unimi.it/apricotmap).
We thus aimed at bridging this gap by organising training and research workshops on PPV with special attention paid to end-users such as fruit producers and nurserymen, in Western and European countries. Leaflets, in nationale language, were produced and distributed to gardening shops and nurseries as well as leaflets describing agronomical, morphological and sensorial quality features of newly produced or/and ready-to-be released, sharka resistant cultivars (see Deliverable . They were transferred to farmers, nurseries and extension services by MARS partners; and uploaded on the MARS website, for further dissemination and printing.
Four types of dissemination activities were performed in the course of MARS, as follows:
• Training workshops on the multiplication and cultivation of PPV resistant apricot material were organised during the first period of MARS, in Spain and Romania, in national languages. Other workshops were organised over the second period, in Spain, Bulgaria, Turkey, Greece, Italy and France. They addressed to fruit producers, managers of major nurseries and advisers from Agricultural Extension Services all over Europe (see all details, copies of the work programme and minutes of the day in the WP3’s second periodic report, available on the MARS collaborative platform). For a complete list of training workshops that took place in the second period, see Table 7 or Deliverable D3.2.
Table 7: MARS training workshops organised by the MARS partners
The MARS leaflet produced in the first period by partners P12 and P06 was translated in national languages and disseminated during their respective training workshops or within their stakeholders’ networks.
• Research Institutions and SMEs participated at meetings and orchard visits under Plant Protection Service and their own organizations, respectively. They were mainly dedicated to PPV control strategies, their effectiveness, their cost-effectiveness, and the end-users perception, including the development of resistant cultivars.
• MARS last dissemination event was organised concomitant to the Fruit Attraction fair, at IFEMA, Madrid, on the 29th of October 2015. Three communications were delivered:
- Welcome and Introduction of the MARS project by Véronique Decroocq (INRA, partner P01)
- The Marker Assisted Selection technology: Legacy of the SharCo FP7 project by Daniele Bassi and Filippo Geuna (UMIL, partner P02)
- The MARS results by Fabienne Moreau (ADNid, partner P10) and Manuel Rubio (CEBAS-CSIC, partner P08)
MARS partners also presented the outcomes of the MARS project in their own breeding or research program, through the display of posters, on site. All communications and posters are available online at www.inra.fr/mars/.
• Publications in peer-reviewed international journals as well as local, vulgarisation newspapers and participation to international symposium and conferences, especially to the World Expo in Milano, where the outcomes of the FP7 SharCo and MARS projects were presented at the EU pavilion, on the 15th of July 2015 (see From SharCo to MARS: A translational research path to solve a major phytosanitary problem http://europa.eu/expo2015/node/731). See the complete list of dissemination tools in the section Use and Dissemination of the Foreground.
- A portal and a database for Marker Assisted Selection of PPV resistant apricot trees
A relational database, based on the 4D (4th Dimension) software has been designed in order to gather and store MARS genotyping data. This database was set up for running under the Windows 2012 Server operating system on an HP Proliant server hosted at the University of Milano (partner P02, UMIL) (see Deliverable D3.3). The database has been fed with genotyping data from the first set of markers developed in SharCo. The process of feeding up the database with data from the second set of markers is still ongoing. Those data are coming from around 25,090 individuals which after screening with the two sets of molecular markers provided 71,608 genotypes. The database is accessible and queryable by the partners and, ultimately (once initial results are suitably published), will be opened to the international community, once the last batch of data is uploaded. An hyperlink will be placed on the international Genome Database for Rosaceae (GDR) (www.rosaceae.org).
List of Websites:
www.inra.fr/mars
Coordinator: decroocq@bordeaux.inra.fr
