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Several Neopestalotiopsis spp. have been reported to affect strawberry worldwide. Here, we sequenced two isolates of Neopestalotiopsis 19-02 (highly virulent) and 13-481 (moderately virulent) recovered from commercial strawberry fields in Florida and generated chromosome-scale assemblies using the Oxford Nanopore Technologies and Illumina platforms. Each genome assembly for 19-02 and 13-481 contained putative telomere sequences at the 5′ and 3′ ends. By analyzing the collinearity of the two newly assembled genomes and verifying the conserved telomere sequence at the end of each chromosome, we confirmed that the genome of Neopestalotiopsis spp. consists of seven basic chromosomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed 98.5% of conserved core genes for both genome assemblies, 19-02 and 13-481. Phylogenetic analysis with internal transcribed spacer, beta-tubulin, and translation elongation factor regions obtained from the assemblies 19-02 and 13-481 demonstrated that the highly virulent isolate 19-02 is phylogenetically distinct from moderate virulent N. rosae 13-481. Comparative genome analysis of the high-quality reference genome assemblies provided in this study will facilitate the identification of genomic regions responsible for the different virulence of the Neopestalotiopsis pathogen to strawberry. [Figure: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Understanding gene function is important for crop improvement and breeding efforts, especially in a genetically complex polyploid plant species such as the octoploid strawberry. Agrobacterium-mediated transient assays are a widely used tool for investigating gene functions and offer a reliable alternative to stable transformation. However, variability in tissue-specific responses and inconsistent applicability of Agrobacterium-mediated transient assay across diverse plant species can be challenging. In this study, we provide a method utilizing Agrobacterium-mediated transient expression to examine the function of genes in octoploid strawberry. Our approach encompasses leaf, root, and fruit tissues, providing a comprehensive strategy for validating gene functions in strawberries. Through meticulous optimization and validation in planta, this method offers valuable insights into gene function in strawberry functional genomics and genetics research. By addressing tissue-specific variability, our methodology serves as a valuable technical resource that could facilitate advancements in identifying gene functions in octoploid strawberry.

Abstract Background The cultivated strawberry (Fragaria × ananassa Duch.) is one of the most economically important horticultural crops worldwide. Botrytis fruit rot (BFR) caused by the necrotrophic fungal pathogen Botrytis cinerea is the most devasting disease of cultivated strawberries. Most commercially grown strawberry varieties are susceptible to BFR, and controlling BFR relies on repeated applications of various fungicides. Despite extensive efforts, breeding for BFR resistance has been unsuccessful, primarily due to lack of information regarding the mechanisms of disease resistance and genetic resources available in strawberry. Results Using a reverse genetics approach, we identified candidate genes associated with BFR resistance and screened Arabidopsis mutants using strawberry isolates of B. cinerea. Among the five Arabidopsis T-DNA knockout lines tested, the mutant line with AtWRKY53 showed the greatest reduction in disease symptoms of BFR against the pathogen. Two genes, FaWRKY29 and FaWRKY64, were identified as orthologs in the latest octoploid strawberry genome, ‘Florida Brilliance’. We performed RNAi-mediated transient assay and found that the disease frequencies were significantly decreased in both FaWRKY29- and FaWRKY64-RNAi fruits of the strawberry cultivar, ‘Florida Brilliance’. Furthermore, our transcriptomic data analysis revealed significant regulation of genes associated with ABA and JA signaling, plant cell wall composition, and ROS in FaWRKY29 or FaWRKY64 knockdown strawberry fruits in response to the pathogen. Conclusion Our study uncovered the foundational role of WRKY transcription factor genes, FaWRKY29 and FaWRKY64, in conferring resistance against B. cinerea. The discovery of susceptibility genes involved in BFR presents significant potential for developing resistance breeding strategies in cultivated strawberries, potentially leveraging CRISPR-based gene editing techniques.

Abstract Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

The fruit skin types of pear (Pyrus spp.) are divided into russet, smooth, and intermediate. One of the important traits in pear breeding programs is russet on pear fruit skin because it affects the commercial value. In the present study, a high-density genetic linkage map of ‘Whangkeumbae’ (smooth) × ‘Minibae’ (russet) was constructed. In addition, quantitative trait loci (QTL) analysis was performed to identify russet related QTL and develop a cleaved amplified polymorphism sequence (CAPS) marker. Together with SNPs derived from Axiom Pear 70K Genotyping Array and genotyping-by-sequencing derived SNPs and SSRs generated in previous study, an integrated genetic linkage map of ‘Whangkeumbae’ × ‘Minibae’ was constructed. A total of 1263 markers were anchored in 17 linkage groups (LGs) with a total genetic distance of 1894.02 cM and an average marker density of 1.48 cM. The chromosome coverage of ‘Whangkeumbae’ × ‘Minibae’ map was improved because the SNPs derived from Axiom Pear 70K Genotyping Array were anchored. QTL analysis was performed using previous russet phenotype data evaluated with russet coverage and Hunter a. As a result of QTL analysis, russet coverage- and Hunter a-related QTLs were identified in LG8 of the ‘Whangkeumbae’ × ‘Minibae’ map, and SNPs located in the QTL region were heterozygous in the ‘Minibae’. Although the russet coverage- and Hunter a-related QTLs were commonly detected in LG8, the logarithm of odds values of SNPs in the QTL region were higher in QTL related to russet coverage than to Hunter a. The CAPS marker (CBp08ca01) was developed using an array SNP located in the russet coverage related QTL, and the genotype of CBp08ca01 showed a 1:1 ratio in ‘Whangkeumbae’ × ‘Minibae’ (χ2 = 0.65, p > 0.05). ‘Whangkeumbae’ and ‘Minibae’ were thought to have rr and Rr genotypes, respectively, and the genetic factors controlling the russet formation might be located in chromosome 8. The CBp08ca01 was able to select F1 individuals with less than 30% russet coverage. Thus, it will be a useful tool for marker-assisted selection in pears.

Colletotrichum crown rot (CCR) caused by Colletotrichum gloeosporioides is a serious threat to the cultivated strawberry (Fragaria × ananassa). Our previous study reported that a major locus, FaRCg1, increases resistance. However, the genomic structure of FaRCg1 and potential candidate genes associated with the resistance remained unknown. Here, we performed comparative transcriptome analyses of resistant ‘Florida Elyana’ and susceptible ‘Strawberry Festival’ after infection and identified candidate genes potentially involved in resistance. In ‘Florida Elyana’, 6,099 genes were differentially expressed in response to C. gloeosporioides. Gene ontology analysis showed that the most upregulated genes were functionally associated with signaling pathways of plant defense responses. Three genes in the genomic region of FaRCg1 were highly upregulated: a von Willebrand Factor A domain-containing protein, a subtilisin-like protease, and a TIFY 11A-like protein. Subgenome-specific markers developed for the candidate genes were tested with a diverse panel of 219 accessions from University of Florida and North Carolina State University breeding programs. Significant and positive associations were found between the high-resolution melting (HRM) marker genotypes and CCR phenotypes. These newly developed subgenome-specific functional markers for FaRCg1 can facilitate development of resistant varieties through marker-assisted selection.

Asian pear scab is a fungal disease caused by Venturia nashicola. The identification of genes conferring scab resistance could facilitate the breeding of disease-resistant cultivars. Therefore, the present study aimed to identify a scab-resistance gene using an interspecific hybrid population ((Pyrus pyrifolia × P. communis) × P. pyrifolia). Artificial inoculation of V. nashicola was carried out for two years. The segregation ratio (1:1) of resistant to susceptible individuals indicated that resistance to V. nashicola was inherited from P. communis and controlled by a single dominant gene. Based on two years phenotypic data with the Kruskal–Wallis test and interval mapping, 12 common markers were significantly associated with scab resistance. A novel scab resistance gene, Rvn3, was mapped in linkage group 6 of the interspecific hybrid pear, and co-linearity between Rvn3 and one of the apple scab resistance genes, Rvi14, was confirmed. Notably, an insertion in pseudo-chromosome 6 of the interspecific hybrid cultivar showed homology with apple scab resistance genes. Hence, the newly discovered Rvn3 was considered an ortholog of the apple scab resistance gene. Since the mapping population used in the present study is a pseudo-BC1 population, pyramiding of multiple resistance genes to pseudo-BC1 could facilitate the breeding of pear cultivars with durable resistance.