Genetics to Breeding

Within the past decade, sweet cherry has become an important Chilean fresh fruit export crop. The cherry fruit industry has the potential to expand and increase the production, quality and volume of exported cherries. A large diversity of commercial sweet cherry cultivars are grown in Chile. In order to determine the genetic diversity and similarity of these germplasms for Sweet Cherry Marker-Assisted Breeding programs, we have created a collection of genomic DNA from 93 commercial sweet cherry cultivars. Sweet cherry (Prunus avium L., 2n=16), is a member of the Prunoideae genus, one of the four Rosaceae subfamilies. Since there is a high degree of synteny between different Prunus species, we have molecularly analyzed this collection of 93 commercial sweet cherry cultivars using 16 SSRs that have been described previously in other Prunus species. The molecular characterization and genetic diversity/similarities among these cultivars will be presented.

This work was funded by FONDEF-Chile grant D04I-1060; 07CN13PBT-167 INNOVA Chile CORFO project; CONICYT Fondecyt 1121021; CONICYT-Regional/GORE O´Higgins/CEAF/ R08I1001; CONICYT Fellowships (21120115 and 24121484 for CK, and 24121618 for VG); Universidad Andrés Bello project DI-78-12/I for KC; and INIA.

Fruit taste depends upon sugars, organic acids, firmness, amino acids, aromatic compounds and so on. Of these components, sugars are one of the most important components of fruit taste. The amount of sucrose, glucose, fructose and sorbitol plays a key role in sweetness of pear fruits. The differences in sugar composition within cultivars exist in Japanese pear. There is much difference in sucrose content and commercially important cultivars accumulate much sucrose. Cultivars with high sucrose accumulation exhibit a rapid increase in sucrose at later stage, while those with low sucrose accumulation exhibit no increase even at later stage. To study whether sucrose content is related to changes in the gene expression of sucrose metabolizing enzymes (sucrose synthase: SUS, soluble acid invertase: AIV and sucrose phosphate synthase: SPS), we examined the expression of five genes such as SUS (PpSUS1), AIV (PpAIV1 and PpAIV2) and SPS (PpSPS1) during development. PpAIV2 is specifically expressed in fruit at later stage by cultivars with low sucrose content. Moreover, we used RFLP analysis to identify PpAIV2 gene linked to sucrose content in ripening fruits. When total DNA was probed with PpAIV2, we identified two bands of 4.4kb and 2.8kb, which were specific to cultivars with low sucrose content. The absence of these bands enabled the identification of high or moderate sucrose content. We have converted these markers in to PCR-based markers. The markers identified here, which are linked to low sucrose content, should be useful for the selection of seedlings with good taste.

We present our findings on genetics of resistance to fire blight (caused by the bacterium Erwinia amylovora) and psylla (Cacopsylla pyri) in a pear inter-specific segregating population between P128R068T003 (Pyrus x bretschneideri X P. communis) and ‘Moonglow’ (P. communis). Asian pears are usually less susceptible to psylla than their European relatives and P128R068T003 was previously demonstrated to be a source of resistance to C. pyri. The other parent, ‘Moonglow’, originated from five P. communis cultivars, and we demonstrated ‘Roi Charles Würtemberg’ to be the source of fire blight resistance. We performed the phenotyping of P128R068T003 x ‘Moonglow’ population at INRA in Angers (France) and detected Quantitative Trait Loci (QTLs) for both fire blight and C. pyri resistance.
Amongst the progenies of this cross, we also observed some instances of hybrid necrosis. This phenomenon is caused by epistatic interactions between genes that have deleterious effects in the hybrid, resulting in dwarfism, tissue necrosis and in some cases lethality. This type of genetic incompatibility has been observed in several plant species and has been known for a long time by plant breeders. We were able to collect leaf samples from the seedlings showing the hybrid necrosis phenotype, scan them with molecular markers using the High Resolution Melting (HRM) technique and identify, for the first time in pear, some of the genomic regions where the lethal genes are located.

Apple scab is a disease caused by the fungus Venturia inequalis which leads to significant economic losses in apple production especially in temperate regions. Breeding programs are attempting to introgress scab resistance genes from wild apple varieties into commercial cultivars for the control of the disease. Most of the commercially available scab resistance varieties to date rely on the Rvi6 (Vf) resistance gene from Malus floribunda 821. The evolution of new pathotypes of V. inequalis, which have caused the breakdown of Rvi6 based resistance at least in northern Europe highlights the need for the characterisation and pyramiding of scab resistance genes from different sources for durable disease resistance. In this study, the scab resistance gene ‘Rvi12’ from Malus baccata ‘Hansen’s baccata #2’ was mapped to apple linkage group 12 in the cross ‘Gala’ × ‘Hansen’s baccata #2’ in an interval between two SSR markers Hi02d05 and Hi07f01. Using the ‘Golden Delicious’ genome sequence, novel SSR markers and SNPs were identified in the Rvi12 mapping interval, and mapped in an extended mapping population of 635 plants. Rvi12 was fine-mapped to an interval of 0.79 cM, spanning 882 kb of the ‘Golden Delicious’ genome sequence. The 23 heterozygous SNPs fine mapped to the Rvi12 mapping interval were screened in eight apple breeding founder lines and for 21 of the 23 SNPs, the allele linked in coupling to the Rvi12 resistance locus were found only in ‘Hansen’s baccata #2’. The SNPs identified will therefore be useful for the efficient identification of apple genotypes carrying the Rvi12 resistance locus.

Strawberry breeding for disease resistance and fruit quality traits is a challenging process due to its complex octoploid genetics. The use of marker-assisted selection (MAS) in plant breeding programs can significantly improve the selection of genotypes through linkage mapping and the identification of quantitative trait loci (QTL) approaches. The success rate of marker development for MAS depends on the complexity, stability and the nature of the QTL, and marker-trait combinations being utilized. In this study, work will be carried out to understand how different complex traits are correlated in the octoploid strawberry at the phenotypic and genotypic level and to investigate how this affects the deployment of markers in breeding. The discovery of the best and easiest traits to phenotype and developing simple ways of capturing phenotypic data in an automated fashion will speed up the development of novel breeding approaches, first through MAS and then genomic selection (GS). A particular focus of current research is on two suites of traits, disease resistance and fruit quality. Work is underway to characterize the nature of Verticillium wilt disease resistance and to uncover and map molecular markers linked to key fruit quality traits such as firmness, sugar levels, fruit size and shape in cultivated strawberry using QTL mapping. An octoploid strawberry mapping population (‘Redgauntlet’ × ‘Hapil’) containing a total of 122 seedlings was used for phenotyping flower-related traits, root architecture and plant characteristics. The existing SSR-based linkage map (‘Redgauntlet’ × ‘Hapil’) will be used for locating genes of interest for MAS and to identify QTLs associated with phenotyped traits.

Neonectria ditissima (formerly Neonectria galligena, anamorph Cylindrocarpon heteronema) is the causal agent of fruit tree canker, which is regarded as a serious economic problem in horticulture. This fungus occurs in a wide range of temperatures but is closely associated with wet weather, and geographic distribution is therefore strongly associated with local climate. Notable damage to apple trees is especially common in some regions like North-Western Europe. Significant efforts Fungicide and culture measures to control the disease usually are not very successful. An alternative approach could be the culture of more resistant cultivars. However, breeding for resistance is hard, resistance tests being time-lasting and labor intensive and resistance being quantitative in nature. Availability of molecular markers for resistance could greatly enhance the prospects of breeding. Currently, nothing is known on the genetic bases of resistance as present in some cultivars. Therefore a focused genomics approach was initiated in order to facilitate the identification of resistance genes. In this study, the transcriptomes of a partially resistant and highly susceptible cultivar are compared for healthy and infected wood. Samples were taken at three different time points, i.e., 5, 15, and 30 days after inoculation.This comparison enabled us to identify a list of candidate genes in response to fungus attack in apple trees. This information will be used in the study of signaling pathways are possibly involved in resistance to fruit tree canker.

Bitter pit is the most economically important physiological disorder affecting apple fruit production. Brown pits develop in the cortical apple flesh – due to the breakdown of the cell plasma membranes – especially during storage, rendering the fruit unmarketable. Environmental conditions and cultural practices play a role in symptom expression, and there is a link between the severity of symptoms and relative concentrations of calcium in the fruit, leading to the application of calcium sprays and dips; however, these are not completely effective. Cultivars vary in susceptibility and thus there is scope for breeding for resistance. In this investigation, we have identified two major QTL controlling bitter pit symptom expression in four large apple mapping populations. The QTL intervals were defined using SSR and SNP markers, and candidate genes from both intervals were characterised. The markers identified will be useful for marker assisted selection in programmes for the genetic improvement of cultivated apple.

Fruit decay occurring during cold storage of apple fruit, e.g., blue mold caused by Penicillium expansum, is a major problem in apple production, especially in organic cultivation and other production systems that do not allow post-harvest anti-fungal treatments. Fruits of susceptible and resistant apple cultivars were challenged with Penicillium expansum spores, and samples of inoculated and control fruits were taken in time series. Differentially expressed genes were identified using an AryANE chip containing 60K apple transcripts. Differentially expressed genes were then analysed for blast similarity, gene ontology and pathway analysis. In another study we quantified some selected chemicals in both inoculated and control fruits, e.g, total polyphenols, individual polyphenolic compounds, total organic acids as well as individual acids to study the possible association of these chemical compounds with the level of fungal resistance. The results of the gene expression study together with the results of the chemical evaluation will enable us to find candidate genes that can be used in breeding programs for development of apple cultivars with improved resistance to storage diseases.

We examined the genetic determinants of vigour control and precocity conferred on a pear scion by Pyrus rootstocks, for the purpose of enabling breeders to develop new pear rootstocks with improved vigour and floral precocity. A segregating population of 450 F1 rootstocks from a P. communis cross between ‘Old Home’ and ‘Louise Bonne de Jersey’ (OHxLBJ), grafted with ‘Doyenne du Comice’ scions, was comprehensively phenotyped for traits related to tree architecture and flowering, and the data modelled in order to describe the growth of the scions. Genotyping was performed using the apple and pear Infinium® II 9K SNP array. A high density SNP-based genetic map was constructed using 546 polymorphic pear and 99 apple markers. QTLs relating to vigour control and precocity were detected on LG5 and LG6 of ‘Old Home’. The LG5 QTL is orthologous to the Dw1 locus from ‘Malling 9’ apple rootstock (Rusholme Pilcher et al. 2008). Analysing the syntenic region linked to this QTL in the P. communis (‘Bartlett’), Malus x domestica (‘Golden Delicious’) and Prunus persica reference genomes should lead to the identification of candidate genes for vigour control in both pear and apple.