First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.

Oleosin Cor a 15 is a novel allergen for Italian hazelnut allergic children.

BACKGROUND: Hazelnut allergy, which is characterized by symptoms that range from mild to severe, is one of the most common allergies in children throughout Europe, and an accurate diagnosis of this allergy is therefore essential. However, lipophilic allergens, such as oleosins, are generally underrepresented in diagnostic tests. We therefore sought to characterize the IgE reactivity of raw and roasted hazelnut oleosins, using the sera of hazelnut-allergic pediatric patients. METHODS: Raw and roasted hazelnut oil body-associated proteins were analyzed by means of 1D and 2D electrophoresis and MS. Oleosin IgE reactivity was assessed by immunoblotting with the sera of 27 children who have confirmed hazelnut allergies and from 10 tolerant subjects. A molecular characterization of the oleosins was performed by interrogating the C. avellana cv. Jefferson and cv. TGL genomes, and through expression and purification of the recombinant new allergen. RESULTS: A proteomic and genomic investigation allowed two new oleosins to be identified, in addition to Cor a 12 and Cor a 13, in hazelnut oil bodies. One of the new oleosins was registered as a new allergen, according to the WHO/IUIS Allergen Nomenclature Subcommittee criteria, and termed Cor a 15. Cor a 15 was the most frequently immunorecognized oleosin in our cohort. Oleosins resulted to be the only immunorecognized allergens in a subgroup of allergic patients who showed low ImmunoCAP assay IgE values and positive OFC and PbP. Hazelnut roasting resulted in an increase in oleosin immunoreactivity. CONCLUSION: A novel hazelnut oleosin, named Cor a 15, has been discovered. Cor a 15 could play a role in eliciting an allergic reaction in a subgroup of pediatric patients that exclusively immunorecognize oleosins. The high prevalence of hazelnut oleosin sensitization here reported further confirms the need to include oleosins in routine diagnostic procedures.

Transcriptome characterization and expression profiling in chestnut cultivars resistant or susceptible to the gall wasp Dryocosmus kuriphilus.

The oriental gall wasp Dryocosmus kuriphilus represents a limiting pest for the European Chestnut (Castanea sativa, Fagaceae) as it creates severe yield losses. The European Chestnut is a deciduous tree, having major social, economic and environmental importance in Southern Europe, covering an area of 2.53 million hectares, including 75,000 ha devoted to fruit production. Cultivars show different susceptibility and very few are resistant to gall wasp. To deeply investigate the plant response and understand which factors can lead the plant to develop or not the gall, the study of transcriptome is basic (fundamental). To date, little transcriptomic information are available for C. sativa species. Hence, we present a de novo assembly of the chestnut transcriptome of the resistant Euro-Japanese hybrid 'Bouche de Bétizac' (BB) and the susceptible cultivar 'Madonna' (M), collecting RNA from buds at different stages of budburst. The two transcriptomes were assembled into 34,081 (BB) and 30,605 (M) unigenes, respectively. The former was used as a reference sequence for further characterization analyses, highlighting the presence of 1444 putative resistance gene analogs (RGAs) and about 1135 unigenes, as putative MiRNA targets. A global quantitative transcriptome profiling comparing the resistant and the susceptible cultivars, in the presence or not of the gall wasp, revealed some GO enrichments as "response to stimulus" (GO:0050896), and "developmental processes" (e.g., post-embryonic development, GO:0009791). Many up-regulated genes appeared to be transcription factors (e.g., RAV1, AP2/ERF, WRKY33) or protein regulators (e.g., RAPTOR1B) and storage proteins (e.g., LEA D29) involved in "post-embryonic development". Our analysis was able to provide a large amount of information, including 7k simple sequence repeat (SSR) and 335k single-nucleotide polymorphism (SNP)/INDEL markers, and generated the first reference unigene catalog for the European Chestnut. The transcriptome data for C. sativa will contribute to understand the genetic basis of the resistance to gall wasp and will provide useful information for next molecular genetic studies of this species and its relatives.

Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins.

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak (Quercus ilex) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 106 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase (pds) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

The de novo, chromosome-level genome assembly of the sweet chestnut (Castanea sativa Mill.) Cv. Marrone Di Chiusa Pesio.

OBJECTIVES: The sweet chestnut Castanea sativa Mill. is the only native Castanea species in Europe, and it is a tree of high economic value that provides appreciated fruits and valuable wood. In this study, we assembled a high-quality nuclear genome of the ancient Italian chestnut variety 'Marrone di Chiusa Pesio' using a combination of Oxford Nanopore Technologies long reads, whole-genome and Omni-C Illumina short reads. DATA DESCRIPTION: The genome was assembled into 238 scaffolds with an N50 size of 21.8 Mb and an N80 size of 7.1 Mb for a total assembled sequence of 750 Mb. The BUSCO assessment revealed that 98.6% of the genome matched the embryophyte dataset, highlighting good completeness of the genetic space. After chromosome-level scaffolding, 12 chromosomes with a total length of 715.8 and 713.0 Mb were constructed for haplotype 1 and haplotype 2, respectively. The repetitive elements represented 37.3% and 37.4% of the total assembled genome in haplotype 1 and haplotype 2, respectively. A total of 57,653 and 58,146 genes were predicted in the two haplotypes, and approximately 73% of the genes were functionally annotated using the EggNOG-mapper. The assembled genome will be a valuable resource and reference for future chestnut breeding and genetic improvement.

Isolation of a gene encoding for a class III peroxidase in female flower of Corylus avellana L.

Hazelnut is a monoecious species characterized by mid-winter blooming and sporophytic incompatibility. The molecular mechanisms at the basis of the female flower development and of the pollen-stigma interaction are little known, although pollination in this species is a critical factor to ensure good yield. Differential display technique was used to study genes expressed during the female flower development, comparing styles before emergence from the bud and styles at full bloom. The full-length cDNA clone, designated CavPrx (Corylus avellana peroxidase) and isolated in mature styles, was characterized as a sequence encoding for a 330 amino acids protein, containing all the conserved features of class III peroxidases. CavPrx resulted expressed only in styles, with a peak in mature styles pollinated with compatible pollen. Class III peroxidases are expressed in several different plant tissue types and are involved in a broad spectrum of physiological processes. Until now, four peroxidases expressed in the stigma were identified in Arabidopsis thaliana and Senecio squalidus: they were assumed to be possibly involved in pollen-pistil interaction, pollen tube penetration/growth and/or in defence against pathogens. CavPrx is the first gene for a floral peroxidase isolated in hazelnut and its expression pattern suggests a possible role in the pollination process.

An In Vitro Protocol for Propagating Castanea sativa Italian Cultivars.

Castanea sativa cv. 'Garrone Rosso' and 'Marrone di Castel del Rio' are two of the most prized varieties in Italy due to their valuable and healthy nuts used for fresh consumption and in the confectionery industry. Despite the growing demand for chestnuts, there are constraints regarding plant propagation that hamper the renewal and new planting of orchards in different areas. Castanea sativa is susceptible to diseases that have caused a reduction in its area of production. For this reason, in vitro culture represents a valuable technique for germplasm preservation and plant multiplication enabling production of a high number of plants for use in breeding programs. Here we present an in vitro micropropagation protocol for Italian Castanea sativa cv. 'Marrone di Castel del Rio' and cv. 'Garrone Rosso' to contribute to the preservation and enhancement of the Italian germplasm. Nodal explants were used as the starting material for in vitro establishment. The cv. 'Marrone di Castel del Rio' showed a high percentage of survival explants (92%) when subjected to long bleach exposure (25 min), in contrast to what was observed for the 'Garrone Rosso' cultivar. Ascorbic acid was found to be the best compound to counteract phenol exudation. The MS3B and DKW media supplied with 0.5 mg/L BAP were effective for in vitro establishment, while the DKW medium (0.1 mg/L BAP and 0.05 mg/L IBA) was preferable for the proliferation phase. A double-layer rooting methodology was used and 35% rooting was observed with 25 mg/L IBA rooting treatment.

Identification of Susceptibility Genes in Castanea sativa and Their Transcription Dynamics following Pathogen Infection.

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed.

First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill.

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications.

Mapping the Genetic Regions Responsible for Key Phenology-Related Traits in the European Hazelnut.

An increasing interest in the cultivation of (European) hazelnut (Corylus avellana) is driving a demand to breed cultivars adapted to non-conventional environments, particularly in the context of incipient climate change. Given that plant phenology is so strongly determined by genotype, a rational approach to support these breeding efforts will be to identify quantitative trait loci (QTLs) and the genes underlying the basis for adaptation. The present study was designed to map QTLs for phenology-related traits, such as the timing of both male and female flowering, dichogamy, and the period required for nuts to reach maturity. The analysis took advantage of an existing linkage map developed from a population of F1 progeny bred from the cross "Tonda Gentile delle Langhe" × "Merveille de Bollwiller," consisting in 11 LG. A total of 42 QTL-harboring regions were identified. Overall, 71 QTLs were detected, 49 on the TGdL map and 22 on the MB map; among these, 21 were classified as major; 13 were detected in at least two of the seasons (stable-major QTL). In detail, 20 QTLs were identified as contributing to the time of male flowering, 15 to time of female flowering, 25 to dichogamy, and 11 to time of nut maturity. LG02 was found to harbor 16 QTLs, while 15 QTLs mapped to LG10 and 14 to LG03. Many of the QTLs were clustered with one another. The major cluster was located on TGdL_02 and consisted of mainly major QTLs governing all the analyzed traits. A search of the key genomic regions revealed 22 candidate genes underlying the set of traits being investigated. Many of them have been described in the literature as involved in processes related to flowering, control of dormancy, budburst, the switch from vegetative to reproductive growth, or the morphogenesis of flowers and seeds.

Whole-genome assembly of Corylus avellana cv "Tonda Gentile delle Langhe" using linked-reads (10X Genomics).

The European hazelnut (Corylus avellana L.; 2n = 2x = 22) is a worldwide economically important tree nut that is cross-pollinated due to sporophytic incompatibility. Therefore, any individual plant is highly heterozygous. Cultivars are clonally propagated using mound layering, rooted suckers, and micropropagation. In recent years, the interest in this crop has increased, due to a growing demand related to the recognized health benefits of nut consumption. C. avellana cv "Tonda Gentile delle Langhe" ("TGdL") is well-known for its high kernel quality, and the premium price paid for this cultivar is an economic benefit for producers in northern Italy. Assembly of a high-quality genome is a difficult task in many plant species because of the high level of heterozygosity. We assembled a chromosome-level genome sequence of "TGdL" with a two-step approach. First, 10X Genomics Chromium Technology was used to create a high-quality sequence, which was then assembled into scaffolds with cv "Tombul" genome as the reference. Eleven pseudomolecules were obtained, corresponding to 11 chromosomes. A total of 11,046 scaffolds remained unplaced, representing 11% of the genome (46,504,161 bp). Gene prediction, performed with Maker-P software, identified 27,791 genes (AED ≤0.4 and 92% of BUSCO completeness), whose function was analyzed with BlastP and InterProScan software. To characterize "TGdL" specific genetic mechanisms, Orthofinder was used to detect orthologs between hazelnut and closely related species. The "TGdL" genome sequence is expected to be a powerful tool to understand hazelnut genetics and allow detection of markers/genes for important traits to be used in targeted breeding programs.

Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation.

Eight transgenic grapevine lines transformed with the coat protein gene of Grapevine fanleaf virus (GFLV-CP) were analyzed for a correlation between transgene expression, siRNAs production and DNA methylation. Bisulphite genome sequencing was used for a comprehensive analysis of DNA methylation. Methylated cytosine residues of CpG and CpNpG sites were detected in the GFLV-CP transgene, in the T7 terminator and in the 35S promoter of three grapevines without transgene expression, but no detectable level of siRNAs was recorded in these lines. The detailed analysis of 8 lines revealed the complex arrangements of T-DNA and integrated binary vector sequences as crucial factors that influence transgene expression. After inoculation with GFLV, no change in the levels of cytosine methylation was observed, but transgenic and untransformed plants produced short siRNAs (21-22 nt) indicating that the grapevine plants responded to GFLV infection by activating a post-transcriptional gene silencing mechanism.

Microsatellite-based genetic relationships in the genus Camellia: potential for improving cultivars.

There is a lack of published microsatellite data that characterizes Camellia spp. To address this, an initial study of sequence tagged microsatellite site (STMS) variation was undertaken with 132 accessions of Camellia spp., which included 24 accessions representing 22 different species or varieties as well as 63 cultivars of C. japonica, 33 cultivars of C. sasanqua, 7 cultivars of C. x vernalis, 3 cultivars of C. x hiemalis, and 2 cultivars of C. hybrida. The four primer sets used (MSCJAF37, MSCJAH46, MSCJAF25, and MSCJAH38) successfully amplified polymorphic alleles in all the species analysed, showing cross-transferability. Overall, 96 alleles were scored. MSCJAH38 primers produced the highest number of bands (30), while MSCJAH46 primers yielded the lowest number (15). The genetic distance between pairs of accessions was estimated on the basis of the Nei coefficient and a principal coordinate analysis was performed. The plot revealed a main differentiation between the C. japonica cultivars and the winter camellias. The distribution of the genetic variation, attributed by AMOVA, particularly highlighted genetic overlap among C. sasanqua cultivars and the cultivars belonging to C. x vernalis, C. x hiemalis, and C. hybrida. In conclusion, this study demonstrated that STMS markers offer a suitable method for detection of genetic variability and molecular study of camellia genotypes.

Development of High-Density Genetic Linkage Maps and Identification of Loci for Chestnut Gall Wasp Resistance in Castanea spp.

Castanea sativa is an important multipurpose species in Europe for nut and timber production as well as for its role in the landscape and in the forest ecosystem. This species has low tolerance to chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu), which is a pest that was accidentally introduced into Europe in early 2000 and devastated forest and orchard trees. Resistance to the gall wasp was found in the hybrid cultivar 'Bouche de Bétizac' (C. sativa × C. crenata) and studied by developing genetic linkage maps using a population derived from a cross between 'Bouche de Bétizac' and the susceptible cultivar 'Madonna' (C. sativa). The high-density genetic maps were constructed using double-digest restriction site-associated DNA-seq and simple sequence repeat markers. The map of 'Bouche de Bétizac' consisted of 1459 loci and spanned 809.6 cM; the map of 'Madonna' consisted of 1089 loci and spanned 753.3 cM. In both maps, 12 linkage groups were identified. A single major QTL was recognized on the 'Bouche de Bétizac' map, explaining up to 67-69% of the phenotypic variance of the resistance trait (Rdk1). The Rdk1 quantitative trait loci (QTL) region included 11 scaffolds and two candidate genes putatively involved in the resistance response were identified. This study will contribute to C. sativa breeding programs and to the study of Rdk1 genes.

A New Sensory Approach Combined with a Text-Mining Tool to Create a Sensory Lexicon and Profile of Monovarietal Apple Juices.

Single-cultivar juices may be a valuable way to introduce different versions of a product to the market and obtain price discrimination. To communicate a product's value, complex characteristics incorporated by each cultivar must be identified. New sensory methods rely on the assessor's ability to recall attributes; however, the use of objective vocabularies may improve the sensory profiling. This work aimed to profile monovarietal apple juices by using projective mapping (PM) combined with ultra-flash profiling (UFP) supported by a sensory wheel built with a text-mining tool. Samples were also analyzed for physicochemical parameters to provide more information to the assessment. The assessor coordinates from PM were used in multiple factor analysis with confidence ellipses to assess differences among samples. A goodness-of-fit test was applied to select the most meaningful descriptors generated through the UFP test by calculating the expected frequency of choosing a descriptor from the sensory wheel and comparing it with the observed values. The methodology provided a more accurate sensory profile compared to previous research on fresh apples and juices. Elstar, Jonagold, and Pinova were considered as sweet juices, and Gravensteiner was described as sour and astringent, with green-apple notes. Rubinette was described as having a strong taste and cloudy aspect.

High density SNP mapping and QTL analysis for time of leaf budburst in Corylus avellana L.

The growing area of European hazelnut (Corylus avellana L.) is increasing, as well as the number of producing countries, and there is a pressing need for new improved cultivars. Hazelnut conventional breeding process is slow, due to the length of juvenile phase and the high heterozygosity level. The development of genetic linkage maps and the identification of molecular markers tightly linked to QTL (quantitative trait loci) of agronomic interest are essential tools for speeding up the selection of seedlings carrying desired traits through marker-assisted selection. The objectives of this study were to enrich a previous linkage map and confirm QTL related to time of leaf budburst, using an F1 population obtained by crossing Tonda Gentile delle Langhe with Merveille de Bollwiller. Genotyping-by-Sequencing was used to identify a total of 9,999 single nucleotide polymorphism markers. Well saturated linkage maps were constructed for each parent using the double pseudo-testcross mapping strategy. A reciprocal translocation was detected in Tonda Gentile delle Langhe between two non-homologous chromosomes. Applying a bioinformatic approach, we were able to disentangle 'pseudo-linkage' between markers, removing markers around the translocation breakpoints and obtain a linear order of the markers for the two chromosomes arms, for each linkage group involved in the translocation. Twenty-nine QTL for time of leaf budburst were identified, including a stably expressed region on LG_02 of the Tonda Gentile delle Langhe map. The stability of these QTL and their coding sequence content indicates promise for the identification of specific chromosomal regions carrying key genes involved in leaf budburst.