The origin and the genetic regulation of the self-compatibility mechanism in clementine (Citrus clementina Hort. ex Tan.).

Self-incompatibility (SI) is a genetic mechanism common in flowering plants to prevent self-fertilization. Among citrus species, several pummelo, mandarin, and mandarin-like accessions show SI behavior. In these species, SI is coupled with a variable degree of parthenocarpy ensuring the production of seedless fruits, a trait that is highly appreciated by consumers. In Citrus, recent evidences have shown the presence of a gametophytic SI system based on S-ribonucleases (S-RNases) ability to impair self-pollen tube growth in the upper/middle part of the style. In the present study, we combined PCR analysis and next-generation sequencing technologies, to define the presence of S7- and S11-Rnases in the S-genotype of the Citrus clementina (Hort. ex Tan.), the self-incompatible 'Comune' clementine and its self-compatible natural mutant 'Monreal'. The reference genome of 'Monreal' clementine is presented for the first time, providing more robust results on the genetic sequence of the newly discovered S7-RNase. SNP discovery analysis coupled with the annotation of the variants detected enabled the identification of 7,781 SNPs effecting 5,661 genes in 'Monreal' compared to the reference genome of C. clementina. Transcriptome analysis of unpollinated pistils at the mature stage from both clementine genotypes revealed the lack of expression of S7-RNase in 'Monreal' suggesting its involvement in the loss of the SI response. RNA-seq analysis followed by gene ontology studies enabled the identification of 2,680 differentially expressed genes (DEGs), a significant number of those is involved in oxidoreductase and transmembrane transport activity. Merging of DNA sequencing and RNA data led to the identification of 164 DEGs characterized by the presence of at least one SNP predicted to induce mutations with a high effect on their amino acid sequence. Among them, four candidate genes referring to two Agamous-like MADS-box proteins, to MYB111 and to MLO-like protein 12 were validated. Moreover, the transcription factor MYB111 appeared to contain a binding site for the 2.0-kb upstream sequences of the S7- and S11-RNase genes. These results provide useful information about the genetic bases of SI indicating that SNPs present in their sequence could be responsible for the differential expression and the regulation of S7-RNase and consequently of the SI mechanism.

Application of new breeding techniques in fruit trees.

Climate change and rapid adaption of invasive pathogens pose a constant pressure on the fruit industry to develop improved varieties. Aiming to accelerate the development of better-adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis, and CRISPR/Cas genome editing hold significant potential for crop trait improvement and have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance and tolerance to abiotic stress and improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. The regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives.

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the 'Made in Italy' label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The 'new genomic techniques' (NGTs), renamed in Italy as 'technologies for assisted evolution' (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific ß-cyclase 2 gene in pigmented citrus fruits.

CRISPR/Cas9 genome editing is a modern biotechnological approach used to improve plant varieties, modifying only one or a few traits of a specific variety. However, this technology cannot be easily used to improve fruit quality traits in citrus, due to the lack of knowledge of key genes, long juvenile stage, and the difficulty regenerating whole plants of specific varieties. Here, we introduce a genome editing approach with the aim of producing citrus plantlets whose fruits contain both lycopene and anthocyanins. Our method employs a dual single guide RNA (sgRNA)-directed genome editing approach to knockout the fruit-specific ß-cyclase 2 gene, responsible for the conversion of lycopene to beta-carotene. The gene is targeted by two sgRNAs simultaneously to create a large deletion, as well as to induce point mutations in both sgRNA targets. The EHA105 strain of Agrobacterium tumefaciens was used to transform five different anthocyanin-pigmented sweet oranges, belonging to the Tarocco and Sanguigno varietal groups, and 'Carrizo' citrange, a citrus rootstock as a model for citrus transformation. Among 58 plantlets sequenced in the target region, 86% of them were successfully edited. The most frequent mutations were deletions (from -1 to -74 nucleotides) and insertions (+1 nucleotide). Moreover, a novel event was identified in six plantlets, consisting of the inversion of the region between the two sgRNAs. For 20 plantlets in which a single mutation occurred, we excluded chimeric events. Plantlets did not show an altered phenotype in vegetative tissues. To the best of our knowledge, this work represents the first example of the use of a genome editing approach to potentially improve qualitative traits of citrus fruit.

A Transcriptional Analysis of the Genes Involved in the Ascorbic Acid Pathways Based on a Comparison of the Juice and Leaves of Navel and Anthocyanin-Rich Sweet Orange Varieties.

Sweet oranges are an important source of ascorbic acid (AsA). In this study, the content of AsA in the juice and leaves of four orange clonal selections, different in terms of maturity time and the presence/absence of anthocyanins, was correlated with the transcription levels of the main genes involved in the biosynthesis, recycling, and degradation pathways. Within each variety, differences in the above pathways and the AsA amount were found between the analysed tissues. Variations were also observed at different stages of fruit development and maturation. At the beginning of fruit development, AsA accumulation was attributable to the synergic action of l-galactose and Myo-inositol, while the l-gulose pathway was predominant between the end of fruit development and the beginning of ripening. In leaves, the l-galactose pathway appeared to play a major role in AsA accumulation, even though higher GalUr isoform expression suggests a synergistic contribution of both pathways in this tissue. In juice, the trend of the AsA content may be related to the decrease in the transcription levels of the GME, GDH, MyoOx, and GalUr12 genes. Newhall was the genotype that accumulated the most AsA. The difference between Newhall and the other varieties seems to be attributable to the GLDH, GalUr12, APX2, and DHAR3 genes.

New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review.

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In the framework of the international scientific community working on fruit tree species, including citrus, NPBTs have mainly been applied to address pathogen threats. Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. To our knowledge, genome editing in citrus via transgenesis has successful for induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1. In the future, NPBTs will also be used to improve fruit traits, making them healthier. The regeneration of plants following the application of NPBTs is a bottleneck, making it necessary to optimize the efficiency of current protocols. The strengths and weaknesses of using explants from young in vitro plantlets, and from mature plants, will be discussed. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. This review aims to summarize methods and approaches available in the literature that are suitable to citrus, focusing on the principles observed before the use of NPBTs.

Recent Advances of In Vitro Culture for the Application of New Breeding Techniques in Citrus.

Citrus is one of the most important fruit crops in the world. This review will discuss the recent findings related to citrus transformation and regeneration protocols of juvenile and adult explants. Despite the many advances that have been made in the last years (including the use of inducible promoters and site-specific recombination systems), transformation efficiency, and regeneration potential still represent a bottleneck in the application of the new breeding techniques in commercial citrus varieties. The influence of genotype, explant type, and other factors affecting the regeneration and transformation of the most used citrus varieties will be described, as well as some examples of how these processes can be applied to improve fruit quality and resistance to various pathogens and pests, including the potential of using genome editing in citrus. The availability of efficient regeneration and transformation protocols, together with the availability of the source of resistance, is made even more important in light of the fast diffusion of emerging diseases, such as Huanglongbing (HLB), which is seriously challenging citriculture worldwide.

Target-Genes Reveal Species and Genotypic Specificity of Anthocyanin Pigmentation in Citrus and Related Genera.

BACKGROUND: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. METHODS: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3'-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. RESULTS: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. CONCLUSION: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

Monitoring the changes in phenolic compounds and carotenoids occurring during fruit development in the tissues of four citrus fruits.

Citrus are fruit crops that are widely cultivated in Southern Italy. The phytochemical profile of citrus depends on the species, the tissue and the ripening stage. This study aimed to perform a comprehensive analysis of the phenolic compounds and carotenoids from four citrus species cultivated in the region of Sicily, Southern Italy, between and within the different tissues and the ripening stages. The selected species were pummelo [C. maxima (Burm. ex Rumph.) Merr.] cv. 'Chandler', lemon [C. limon (L.) Burm. f] cv. 'Akragas', and sweet orange [C. sinensis (L.) Osbeck] cvs 'Tarocco' and 'Washington navel'. Fruits were harvested four times every five weeks from Sep 2018 to Jan 2019. At any stage, flavedo and albedo showed the highest contents of phytochemicals. Elevated levels of phenolic compounds, e.g., narirutin, were found in the flavedo of unripe lemon and 'Tarocco' orange (54.8 ± 8.15 and 248 ± 30.3 mg kg-1 dw, respectively). Narirutin decreased significantly (p < 0.05) during maturity. An analogous trend (p < 0.05) was observed in the albedos of unripe pummelo and 'Washington navel' orange for vanillic acid (47.7 ± 8.31 and 54.7 ± 9.06 mg kg-1 dw, respectively). Phenolics decreased during maturity in the juice sacs as well, apart from lemon, e.g., hesperidin peaked at the last developmental stage (2213 ± 173 mg kg-1 dw; p < 0.0001). With regard to carotenoids, flavedos were the richest tissues. In general, flavedos increased the levels of carotenoids during ripening, apart from pummelo in which cumulative carotenoids were the highest in September (135 ± 16.5 mg kg-1 dw). Violaxanthin was the main carotenoid of the juice sacs, and was abundant in ripe 'Washington navel' orange (9.09 ± 1.73 mg kg-1 dw in Jan). The present investigation delivers valuable information for the comprehensive utilization of citrus fruits from Southern Italy by the local food industry.

Noemi Controls Production of Flavonoid Pigments and Fruit Acidity and Illustrates the Domestication Routes of Modern Citrus Varieties.

In citrus, the production of anthocyanin pigments requires the activity of the transcriptional activator Ruby. Consequently, loss-of-function mutations in Ruby result in an anthocyaninless phenotype [1]. Several citrus accessions, however, have lost the ability to produce these pigments despite the presence of wild-type Ruby alleles. These specific mutants have captivated the interest of botanists and breeders for centuries because the lack of anthocyanins in young leaves and flowers is also associated with a lack of proanthocyanidins in seeds and, most notably, with an extreme reduction in fruit acidity (involving about a three-unit change in pH). These mutants have been defined collectively as "acidless" [2-4]. We have identified Noemi, which encodes a basic helix-loop-helix (bHLH) transcription factor and which controls these apparently unrelated processes. In accessions of Citron, limetta, sweet lime, lemon, and sweet orange, acidless phenotypes are associated with large deletions or insertions of retrotransposons in the Noemi gene. In two accessions of limetta, a change in the core promoter region of Noemi is associated with reduced expression and increased pH of juice, indicating that Noemi is a major determinant of fruit acidity. The characterization of the Noemi locus in a number of varieties of Citron indicates that one specific mutation is ancient. The presence of this allele in Chinese fingered Citrons and in those used in the Sukkot Jewish ritual [5] illuminates the path of domestication of Citron, the first citrus species to be cultivated in the Mediterranean. This allele has been inherited in Citron-derived hybrids with long histories of cultivation.

Changes in Anthocyanin Production during Domestication of Citrus.

Mandarin (Citrus reticulata), citron (Citrus medica), and pummelo (Citrus maxima) are important species of the genus Citrus and parents of the interspecific hybrids that constitute the most familiar commercial varieties of Citrus: sweet orange, sour orange, clementine, lemon, lime, and grapefruit. Citron produces anthocyanins in its young leaves and flowers, as do species in genera closely related to Citrus, but mandarins do not, and pummelo varieties that produce anthocyanins have not been reported. We investigated the activity of the Ruby gene, which encodes a MYB transcription factor controlling anthocyanin biosynthesis, in different accessions of a range of Citrus species and in domesticated cultivars. A white mutant of lemon lacks functional alleles of Ruby, demonstrating that Ruby plays an essential role in anthocyanin production in Citrus Almost all the natural variation in pigmentation by anthocyanins in Citrus species can be explained by differences in activity of the Ruby gene, caused by point mutations and deletions and insertions of transposable elements. Comparison of the allelic constitution of Ruby in different species and cultivars also helps to clarify many of the taxonomic relationships in different species of Citrus, confirms the derivation of commercial varieties during domestication, elucidates the relationships within the subgenus Papeda, and allows a new genetic classification of mandarins.

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain.

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed. The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.

Involvement of a citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation.

BACKGROUND: Transposable-element mediated chromosomal rearrangements require the involvement of two transposons and two double-strand breaks (DSB) located in close proximity. In radiobiology, DSB proximity is also a major factor contributing to rearrangements. However, the whole issue of DSB proximity remains virtually unexplored. RESULTS: Based on DNA sequencing analysis we show that the genomes of 2 derived mutations, Arrufatina (sport) and Nero (irradiation), share a similar 2 Mb deletion of chromosome 3. A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5' end of the deletion. The Arrufatina Mule displayed "dissimilar" 9-bp target site duplications separated by 2 Mb. Fine-scale single nucleotide variant analyses of the deleted fragments identified a TTC-repeat sequence motif located in the center of the deletion responsible of a meiotic crossover detected in the citrus reference genome. CONCLUSIONS: Taken together, this information is compatible with the proposal that in both mutants, the TTC-repeat motif formed a triplex DNA structure generating a loop that brought in close proximity the originally distinct reactive ends. In Arrufatina, the loop brought the Mule ends nearby the 2 distinct insertion target sites and the inverted insertion of the transposable element between these target sites provoked the release of the in-between fragment. This proposal requires the involvement of a unique transposon and sheds light on the unresolved question of how two distinct sites become located in close proximity. These observations confer a crucial role to the TTC-repeats in fundamental plant processes as meiotic recombination and chromosomal rearrangements.

Characterization of the glutathione S-transferase gene family through ESTs and expression analyses within common and pigmented cultivars of Citrus sinensis (L.) Osbeck.

BACKGROUND: Glutathione S-transferases (GSTs) represent a ubiquitous gene family encoding detoxification enzymes able to recognize reactive electrophilic xenobiotic molecules as well as compounds of endogenous origin. Anthocyanin pigments require GSTs for their transport into the vacuole since their cytoplasmic retention is toxic to the cell. Anthocyanin accumulation in Citrus sinensis (L.) Osbeck fruit flesh determines different phenotypes affecting the typical pigmentation of Sicilian blood oranges. In this paper we describe: i) the characterization of the GST gene family in C. sinensis through a systematic EST analysis; ii) the validation of the EST assembly by exploiting the genome sequences of C. sinensis and C. clementina and their genome annotations; iii) GST gene expression profiling in six tissues/organs and in two different sweet orange cultivars, Cadenera (common) and Moro (pigmented). RESULTS: We identified 61 GST transcripts, described the full- or partial-length nature of the sequences and assigned to each sequence the GST class membership exploiting a comparative approach and the classification scheme proposed for plant species. A total of 23 full-length sequences were defined. Fifty-four of the 61 transcripts were successfully aligned to the C. sinensis and C. clementina genomes. Tissue specific expression profiling demonstrated that the expression of some GST transcripts was 'tissue-affected' and cultivar specific. A comparative analysis of C. sinensis GSTs with those from other plant species was also considered. Data from the current analysis are accessible at http://biosrv.cab.unina.it/citrusGST/, with the aim to provide a reference resource for C. sinensis GSTs. CONCLUSIONS: This study aimed at the characterization of the GST gene family in C. sinensis. Based on expression patterns from two different cultivars and on sequence-comparative analyses, we also highlighted that two sequences, a Phi class GST and a Mapeg class GST, could be involved in the conjugation of anthocyanin pigments and in their transport into the vacuole, specifically in fruit flesh of the pigmented cultivar.

Root protein profiles of two citrus rootstocks grown under iron sufficiency/deficiency conditions.

Two citrus rootstocks, one sensitive to iron deficiency (Swingle Citrumelo (SCO)) and the other tolerant (Carrizo Citrange, (CC)), were studied to characterize variation in their root protein profile induced by iron-deficient conditions. Plants of both rootstocks were grown in two different soils, one volcanic (v) and the other calcareous (c), containing 0% and 10% active Lime, respectively. To evaluate the effects of the calcareous soil on the protein accumulation of both rootstocks, the root protein profiles (SCc vs. SCv and CCc vs. CCv) were characterized by two-dimensional gel electrophoresis, thus obtaining, for the first time, a reference map of this previously uncharacterized proteome. A total of 219 spots, significantly changed in abundance, were analyzed by high-performance Liquid chromatography nano electrospray ionization tandem mass spectrometry. The identified proteins were classified according to their putative function and known biosynthetic pathways. Principal component analysis, comparing the four sets of data, indicated that each group clustered together with low variance and that CCv and CCc data sets were well differentiated, whereas SCv and SCc were similar.

Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges.

Traditionally, Sicilian blood oranges (Citrus sinensis) have been associated with cardiovascular health, and consumption has been shown to prevent obesity in mice fed a high-fat diet. Despite increasing consumer interest in these health-promoting attributes, production of blood oranges remains unreliable due largely to a dependency on cold for full color formation. We show that Sicilian blood orange arose by insertion of a Copia-like retrotransposon adjacent to a gene encoding Ruby, a MYB transcriptional activator of anthocyanin production. The retrotransposon controls Ruby expression, and cold dependency reflects the induction of the retroelement by stress. A blood orange of Chinese origin results from an independent insertion of a similar retrotransposon, and color formation in its fruit is also cold dependent. Our results suggest that transposition and recombination of retroelements are likely important sources of variation in Citrus.

Use of a custom array to study differentially expressed genes during blood orange (Citrus sinensis L. Osbeck) ripening.

A flesh-specific oligonucleotide custom array was designed to study gene expression during blood orange ripening. The array included 301 probes derived from a subtracted SSH library, a cDNA-AFLP collection, and a set of regulatory genes from the Harvest citrus database. The custom array was hybridized using samples of Moro, a pigmented cultivar, and Cadenera, a common cultivar, at three different ripening stages: the immature phase, the halfway point of maturation (corresponding to the start of Moro pigmentation) and the full ripening. Of the 301 probes, 27 in total, corresponding to 20 different transcripts, indicated differential expression in stage-to-stage and/or cultivar-to-cultivar comparisons. Transcripts encoding for anthocyanin biosynthesis represented most of the total over-expressed probes. The remaining differentially expressed transcripts were functionally associated with primary metabolism as flavor biosynthesis, defense and signal transduction. The expressed products associated with probes indicating differential expression were confirmed by qRT-PCR. The microarray was designed considering a small collection of sequences useful for monitoring specific pathways and regulatory genes related to fruit ripening and anthocyanin pigmentation. The main novelty of this customization is the use of expressed sequences specifically derived from blood orange flesh to study different cultivars and ripening stages, and the provision of further information about processes related to anthocyanin pigmentation in citrus fruit flesh.