Abstract
A major Sharka resistance determinant (PPVres) has been identified on chromosome 1 in apricot. Following the comparison of the ‘Resistant’ and ‘Susceptible’ haplotypes of the PPVres region, some candidate genes have been identified. The project develops over the following main objectives: (a) development of a dense linkage map and QTL analysis on a large available progeny; (b) identification of candidate genes and markers tightly associated in a panel of resistant genotypes; (c) combined analysis of ‘Lito’ transcriptome and small RNAs to disclose the differential gene expression and silencing effect during PPV infection; (d) validation of candidate genes by genetic transformation, genome editing and virus-induced gene silencing. Outlines of the main project results The PRIN22 research project was focused on understanding and combating Sharka disease in apricot trees through genetic and biotechnological methods and herewith are summarized the main project achievements. 1. A high-resolution genetic map of the ‘Lito’ x ‘BO81604311’ progeny was produced by using a SNP panel based on a GBS approach. A high coverage whole genome sequencing (40X) of the parental lines was performed for variant calling by using an improved haplotype-resolved genomic sequence of the ‘Lito’ parent. After a filtering step, SNP segregating in ‘Lito’ and in ‘BO81604311’ were used for the two maps construction. The molecular map of Lito is covering 889 cM while that of BO81604311 is 1378 cM. Both maps have a very good genome coverage with markers well distributed along the chromosomes. Only a few gaps have been identified in some chromosomes. Nevertheless, the maps resulted in good quality for QTL analysis. These mapping data are also under further scrutiny in order to make refinements of the assembly of the available Lito genome sequence. The high-resolution genetic map produced appears to be the most detailed available and is a valuable source of new genome-wide markers in apricot. 2) QTL analysis. The sharka resistance data, checked and implemented for both the M and D strains, were used for QTL analysis by MapQTL, considering both the maximum level of susceptibility of each plant and the recovery observed after three years of observation (as reported in Dondini et al., 2011). The QTL analysis was performed with both the Kruskall Wallis and Interval mapping (mixture model) algorithm. The analysis confirmed a very strong QTL on chromosome 1 of ‘Lito’. The QTL peaks on the Chr1_8555222 SNP marker independently from the resistance dataset used in the analysis. Minor QTLs peaks were also identified in other regions of chromosome 1 that resulted specific for the M strain. Minor QTLs for the different resistance datasets were also observed in other chromosomes but with a significance level just above the threshold established by the permutation test. In detail, for the M strain on chr8 for the dataset with recovery and chr 6 for the maximum susceptibility dataset. For the D strain a minor QTL has been identified in Chr3 with the resistance dataset considering the recovery. The identified QTLs were aligned to the newly available haplotype-resolved ‘Lito’ genome sequence to identify candidate genes of resistance. 3. Structural Characterization of PPV High-resolution cryo-electron microscopy (2.9 Å) revealed the filamentous architecture of the PPV-M virion, confirming a conserved potyvirus coat protein (CP) organization with a single-stranded RNA genome. The N-terminal CP region was shown to be intrinsically disordered and rich in post-translational modifications, predominantly phosphorylation, consistent with predicted amyloidogenic properties. PPV was successfully propagated in Nicotiana benthamiana, purified, and analyzed by electron microscopy and Western blotting. Average virus yield reached approximately 0.8 mg per 100 g of tissue. Purified viral RNA was used to establish a sensitive RT-qPCR standard curve (R² = 0.98), defining a detection limit of 1 pg of viral RNA (Cq 38.6). 4. Biolistic Transmission of PPV Purified PPV RNA was delivered by gene gun into N. benthamiana, apricot cv. ‘Lito’, and myrobalan 29C. In N. benthamiana, 40% of plants became infected and displayed an unusual, more severe symptom phenotype than the parental isolate. The altered phenotype was stable after reinoculation, suggesting selection of viral variants during biolistic transmission. In woody hosts, infection was confirmed by RT-qPCR in 43% of myrobalan and 60% of apricot plants two months after inoculation. 5. Host Response and Resistance Studies in Apricot ‘Lito’ Expression analyses of defense-related genes in detached leaves challenged with viruliferous aphids revealed transient induction of Pa31, PR-1, and PAL2, while virus accumulation remained low. RNA-seq results corroborated RT-qPCR data, showing modest but measurable transcriptional changes following infection, with low viral RNA abundance. These findings suggest that ‘Lito’ supports limited virus replication and activates a restricted defense response. 6. PPV Transmission and Grafting Experiments Aphid-mediated inoculation resulted in successful infection of one out of four myrobalan 29C plants, confirming susceptibility at low viral titers. In grafting experiments, PPV-infected myrobalan rootstocks were grafted with ‘Lito’ scions; two scions became infected, demonstrating virus movement across the graft union. Samples from infected and healthy grafts are being prepared for PacBio long-read sequencing to generate reference transcriptomes of healthy and PPV-infected ‘Lito’. 7. Development of PNRSV-Based Viral Vectors Full-length infectious PNRSV clones carrying inserts targeting PDS and Pa31 genes were engineered and delivered by biolistics. In cucumber, all constructs replicated systemically, as confirmed by EM and molecular assays, although only the wild-type construct produced visible symptoms. Lower RNA accumulation in insert-bearing constructs suggests activation of silencing mechanisms. Comparable constructs were also introduced into myrobalan and apricot ‘Lito’; infection was confirmed in several plants by RT-qPCR, and long-term monitoring is ongoing. 8. Construct preparation. A set of constructs for both genetic transformation and DNA-editing were designed, these constructs include members of the MATH-domain gene family (Pa34 and Pa31) and GFS12 (a BEACH-domain protein). These constructs are ready to be used for apricot transformation once the transformation protocol will be available. 9. Apricot Regeneration and transformation A strong cultivar effect was observed regarding micropropagation ability. Apricot somatic tissues confirmed to be very recalcitrant to regeneration despite the various tested media. For this reason, the regeneration ability of embryonic tissues was tested to try to improve transformation efficiency. Seeds from resistant and susceptible cultivars were used and a system to take into account the seed variability was settled and tested: cotyledons of each seed were used for genetic transformation while the corresponding embryo was separately cultured in vitro to be used as negative control. An Agrobacterium-mediated protocol was used on cotyledon with a standard construct as proof of concept. Despite the low regeneration efficiency, two independent GM lines were obtained from cotyledon tissues of ‘Reale di Imola’. These cotyledon-derived lines resulted positive for the nptII gene but not the corresponding embryos. The project achieved: - Development of a large panel of SNPs markers by a GBS approach on a large segregating progeny (LxB progeny of 341 plants); - Implementation of the molecular map of the LxB cross and QTL analysis by using an implemented sharka resistance dataset in order to identify small effect QTLs; - Optimized protocol for virus purification from herbaceous test plant (Nicotiana benthamiana) for genomic RNA extraction and high-resolution structural elucidation of PPV-M virions; - Proof-of-concept demonstration of the PPV viral RNA biolistic inoculation method (Helios gene gun) in host plants; - Establishment of a quantitative RT-qPCR detection system. - Inoculation of PPV by viruliferous aphids (Myzus persicae) in apricot detached leaf (cv Lito) to assess the early response to viral infection through transcriptomic and differential gene expression analysis; - Biolistic inoculation of PNRSV infectious transcripts and silencing constructs in herbaceous and woody hosts; - Grafting of resistant apricot (cv Lito) budwood on PPV-infected and healthy myrobalan 29C rootstock to apply for accurate transcriptomic analysis (long reads Pac-Bio); - Implementation of the in vitro culture protocols in apricot to enhance propagation and genetic modification; - Identification of candidate genes for sharka resistance and construct preparation; - Production and use of a PNRSV-derived Virus-Induced Gene Silencing (VIGS) platform in apricot for functional validation of candidate genes. - Transcriptomic and deep-sequencing analyses to further clarify host-virus interactions and viral adaptation mechanisms.
Dettagli del progetto
Responsabile scientifico: Stefano Tartarini
Strutture Unibo coinvolte:
Dipartimento di Scienze e Tecnologie Agro-Alimentari
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 206.194,00
Contributo totale Unibo: Euro (EUR) 74.330,00
Durata del progetto in mesi: 24
Data di inizio
12/10/2023
Data di fine:
28/02/2026
