The nature of resistance to Neonectria ditissima in apple species

In the UK, land devoted to modern apple production is rising at a rate of between 7 and 10% per annum, however (similar to the global situation) the combination of more intensive planting of canker-susceptible cultivars has led to an increase in losses due to the fungal pathogen Neonectria ditissima. Problems in both in-field production (tree death and yield reduction) and post-harvest losses (due to postharvest rot), call for an innovative approach to combat this disease.

Approximately 50% of apples produced in the UK are cultivars derived from two progenitor apple varieties, Cox and Gala, both of which are susceptible to fungal canker, indeed these newer varieties such as Kanzi, Rubens and Jazz are extremely susceptible to canker, which has led to large problems in orchard establishment and increasingly post-harvest losses, especially in wet years. In severe cases, in field yield decline can reduce potential pre-harvest yield by at least 25% due to loss of fruiting wood and 5% of postharvest apple crops are lost due to Neonectria infection. There is poor chemical control of canker of trees due to its systemic nature of the disease. It has been shown that the pathogen is often present in the plants from a young age (in the nursery) and that both the rootstock and the scion can be infected, but remain asymptomatic for long periods of time.

Natural resistance to canker has been well documented, but extremely poorly deployed in modern rootstock or scion breeding programmes. The reasons for this can be attributed to the complex nature of resistance (making selection without molecular markers difficult), the cost of mass screening to eliminate highly susceptible individuals, different types of resistance (all of which are uncharacterised), the relatively recent re-emergence of the problem and the potentially heterogeneous nature of the pathogen.

This proposal will use cutting-edge genomics approaches to identify natural plant-derived resistance to this important pathogen in both scion varieties and in rootstocks. In this project, a multi-parental population will be used to characterise resistance and a combination of targeted genome sequencing techniques and pedigree-based genotyping will be used to accurately narrow down the specific genes responsible for resistance. Once obtained, in proof of principle experiments, genes underpinning the resistance will be transferred into a susceptible cultivar, in order to validate the resistance in other genetic backgrounds.

The industry are supporting this proposal through the LINK scheme, as they recognise the need for a comprehensive strategy to identify the genetic basis of disease resistance, for use in marker assisted breeding (MAB) and other breeding techniques.

Understanding the genetic basis of resistance to fungal canker is important due to the need for resistant cultivars for global production. In order to fulful this requirement a programme of research querying quantitative resistance in the cultivated apple and species-level resistance in apple roostocks is proposed. Apple (Malus x domestica) is an outcrossing species with a highly heterozygous genome. Using the latest, PCR-free library preparation techniques, along with cutting-edge genome assemblers (DISCOVAR) founding parents of a multi-parental set of lines and mapping population parents will be sequenced. Although QTL mapping will be unbiased, in order to accurately capture the highly variable resistance gene space, (for this and for other disease resistance studies), both NB-LRR and eLRR families will be queried using a PacBio-RenSeq approach to resolve phased candidate resistance genes, without recourse to a BAC library. Genotyping the multiparental population using 20k illumina SNP chips (and the rootstock population with GbS) will allow full imputation of genome sequences onto individual clones. Phenotyping replicated progenies, using a variety of pathogenicity tests, from cut shoot tests, fruit tests and whole tree infection in multiple environments will allow QTL mapping to be carried out to identify the genetic basis of resistance. Further analysis of RNAseq data of infection will allow the role of different candidate genes underlying high resolution QTL to be studied. Validation of single and multiple candidate genes using multi-gene GoldenGate constructs transformed into susceptible lines will allow validation of QTL; promoter analysis will elucidate tissue specificity of resistance responses. This will also facilitate further characterisation of candidate pathogenicity genes in Neonectria, from ongoing collaborative pathogen genome sequencing work.

This grant will have a global impact, both on the research field internationally and on the international industry, especially the UK industry. Through full engagement with industry stakeholders, maximum translation of this research will be ensured, driving forward the UK plant breeding industry in a globally competitive market.

Direct beneficiaries:

1. Commercial private sector
The UK and international plant breeding sector will benefit enormously from this endeavour and will allow these industries to first develop better markers for QTL (most traits in apple are quantitative and multiple QTL underpin these) and move from marker level associations to candidate gene associations. This is important for next-generation genome editing approaches and functional validation of candidate genes. This moves the industry very quickly to a point where pedigree-based selection and genome-wide selection are affordable and tractable options for crop improvement. Placing this in the hands of the UK partners will give the UK business a significant competitive edge.

2. Fruit growing sector in the UK
UK industry will benefit as they will be able to access a resource that is beyond their means to create. Longer term it is anticipated that the UK partners will make significant use of this resource and knowledge generated from this pre-competitive work. This may lead to further competitive work funded by other research bodies (e.g. innovate UK or AHDB). Advancing genomic resources in horticultural crops is a key aim of the AHDB-Horticulture and evidenced by their support in this proposal (Benefit within 5-10 years).

3. Public and retail sector
Several UK retailers aim to double sales of UK-produced fruit by 2020; this project will assist that aim and improve UK productivity and competitiveness. Downstream science conducted utilising the resources generated in this project will lead to more reliable production methods and potentially reduce wastage in the supply chain (through reduced inputs and better variety development) (Benefit within 5-10 years).

Indirect beneficiaries
The wider apple growing industry (UK and beyond)
As a result of resistance markers to fungal canker, the rate of change of varietal development will increase, leading to greater benefits to downstream growers, packers and producers.

Government, public and policy benefits
The public will benefit, not only from the improved position of UK agribusiness (and access of breeders to novel technologies), but also through the long term improvement in supply chain resilience through improved cultivar development. In the longer term the public will benefit through increased food security and sustainability, as a result of scientific improvements on horticultural crops. This feeds in to many UK Government and EU policy agendas including: health (improving produce quality, pesticides (reducing residues through improved resistance), water (ability to grow nearer water courses), climate (growing crops perennially will improve carbon sequestration) and environment (reduced carbon and pesticides) (Benefit within 5-10 years).