Diverse Citrus Research Articles

Genome-wide investigation of glycoside hydrolase 9 (GH9) gene family unveils implications in orchestrating the mastication trait of Citrus sinensis fruits

Mastication trait of citrus significantly influences the fruit’s overall quality and consumer preference. The accumulation of cellulose in fruits significantly impacts the mastication trait of citrus fruits, and the glycoside hydrolase 9 (GH9) family plays a crucial role in cellulose metabolism. In this study, we successfully identified 32 GH9 genes from the Citrus sinensis genome and subsequently conducted detailed bioinformatics analyses of the GH9 family. Additionally, we profiled the spatiotemporal expression patterns of CsGH9 genes across four distinct fruit tissue types and six crucial developmental stages of citrus fruits, leveraging transcriptome data. Parallel to this, we undertook a comparative analysis of transcriptome profiles and cellulose content among diverse fruit tissues spanning six developmental stages. Furthermore, to identify the pivotal genes involved in cellulose metabolism within the GH9 family during fruit maturity, we employed correlation analysis between cellulose content and gene expression in varying tissues across diverse citrus varieties. This analysis highlighted key genes such as CsGH9A2/6 and CsGH9B12/13/14/22. Collectively, this study provides an in-depth analysis of the GH9 gene family in citrus and offers novel molecular insights into the underlying mechanisms governing the mastication trait formation in citrus fruits.

DIVERSITY OF Citrus spp. FROM PENAJAM PASER UTARA, EAST KALIMANTAN BASED ON MORPHOLOGICAL CHARACTERS

ARTICLE HIGLIGHTS- This research is the first study to provide data on the diversity of Citrus spp. from North Penajam Paser Regency (IKN) area, East Kalimantan for further development and breeding program. - This research offers new information on the distribution of Citrus spp. from the North Penajam Paser Regency (IKN) area, East Kalimantan. - This research provides information on the phenetic relationships among Citrus spp. from the North Penajam Paser Regency (IKN) area, East Kalimantan, and the morphological characteristics that play the most significant role in their clustering. ABSTRACTCitrus spp. is local Indonesian fruit with high diversity and widely distributed around Indonesia country. Diversity and relationship data of Citrus spp. from Penajam Paser Utara-East Kalimantan has never been studied before. This study aimed to provide the diversity and relationship data of Citrus spp. from Penajam Paser Utara using morphological characteristics which served as the basic data for further development and breeding. The research was conducted through observation and sampling, followed by characterization and scoring of morphological data, and lastly the data analysis. The research succeeded in finding 35 accessions of Citrus spp. consisting of C. limon “Jeruk Lemon”, C. suhuensis, “Jeruk Siam”, C. aurantiifolia “Jeruk Nipis”, C. sinensis “Jeruk Manis”, C. maxima “Jeruk Bali”, C. microcarpa “Jeruk Kalamansi”, and Citrus sp. "Jeruk Sunkist" spread in sub-district Penajam, Sepaku, Babulu, and Waru. The dendrogram to clustering Citrus spp. in PPU was divided into two large clusters. Cluster A was grouped based on the character of tree habitus, cordate leaf apex, entire leaf margin, pink mesocarp color, and seed absence. Cluster B was grouped based on the character of the shrubs habitus, acute and rounded leaf apex, crenate leaf margin, white to orange mesocarp color, and numerous seeds in the fruit. This diversity made Citrus spp. from Penajam Paser Utara had high potential value for further development through a plant breeding program.

Variability analysis of the Phenotypic Characteristics of some Grapefruit and Pomelo under Moroccan Conditions

Citrus is an economically important fruit crop with a long history of cultivation worldwide, and many citrus varieties are grown extensively in Morocco, both for domestic consumption and export. Nevertheless, the genetic diversity of this genus in Morocco has not been reported upon. Our objective was to explore phenotypic variations in grapefruit and pomelo genotypes for targeted breeding, enhancing key traits, and identifying opportunities for genetic improvement. We emphasize practical implications and outline future research directions for developing improved citrus varieties. This present study physiochemically analyzes eight grapefruit (Citrus paradisi) and two pomelo (Citrus maxima) cultivars to assess the genetic variability of these fruits’ physicochemical characteristics. The results revealed significant variance among the genotypes for all traits. For example, the phenotypic coefficient of variation (PCV) was higher than genotypic coefficient of variation (GCV) for all the traits. Moreover, a high degree of heritability was recorded for seed number, β-carotene, vitamin C, juice yield (%), total soluble solids, titratable acidity, maturity index, fruit diameter, and fruit weight, whereas limited heritability was observed for fruit length, peel thickness, and segment number per fruit. The high genetic advance scores corresponded to the high degree of heritability estimates for seed number per fruit, β-carotene, juice yield, and vitamin C, suggesting the existence of additive genetic effects. Our principal component analysis (PCA) identified four components with cumulative variance of 93.20%, while hierarchical cluster analysis grouped the collection into five clusters. In conclusion, phenotypic distinctions among grapefruit and pomelo genotypes reveal opportunities for genetic enhancement through selection or hybridization. Varied traits, including seed count, vitamin C, beta-carotene, and juice yield, across genotypes suggest potential avenues for targeted breeding and practical applications in diverse citrus varieties.

Ethnobotany and diversity of Citrus spp. (Rutaceae) as a source of “Kem-kem” traditional medicine used among the Karo sub-ethnic in North Sumatra, Indonesia

The Karo sub-ethnic is one of five Batak sub-ethnicities in the Karo Regency, North Sumatra Province, Indonesia. They are famous for their local knowledge about the traditional use of medicinal plants to treat various diseases. The “Kem-kem” traditional medicine is one of the traditional healing practices that involve using plants passed down through generations from their ancestors. One of the plant genus group in the Rutaceae family utilized in the traditional “Kem-kem” healing practice is a citrus known as “Rimo”. This study aims to document the local knowledge about the diversity of Citrus spp. as Kem-kem's herbal medicinal plant. This study was conducted from April to July 2023 in the Kabanjahe and Berastagi districts, Karo Regency, North Sumatra. Data was collected using interviews with traditional healers, herbal medicine vendors, and direct observations at traditional markets, involving a total of 8 Citrus spp. The Karo uses “Rimo” with different local names as sources of traditional medicinal ingredients in practicing “Kem-kem”. There are 15 local names comprising eight species of Citrus. Four are hybrids, i.e., Citrus x aurantiifolia (Christm.) Swingle, Citrus × aurantium L, Citrus × junos Siebold ex Yu.Tanaka, and Citrus × taitensis Risso. Two of the remaining species are recognized in infraspecific rank, one variety (Citrus medica var. sarcodactylis (Hoola van Nooten) Swingle) and one form (Citrus × aurantium f. deliciosa (Ten.) M.Hiroe). They were used as material sources for Kem-kem traditional medicine to treat at least nine health problems. There are two species with six local names included in the Least Concern (LC) category, namely C. medica (Rimo Gawang, Rimo Hantuantu, Rimo Kayu), C. medica var. sarcodactylis (Rimo Kuku Harimau), and C. medica (Rimo Telur Buaya), C. maxima (Burm.) Merr. (Rimo Malem). Nine local names are included in the Not Evaluated (NE) category, namely C. × junos (Rimo Kejaren), C. × taitensis (Rimo Jungga), C. × aurantium f. deliciosa (Rimo Keling), C. × aurantium (Rimo Kersik), Citrus hystrix DC. (Rimo Mukur), C. × taitensis (Rimo Puraga), C. × aurantium (Rimo Kalele), Citrus swinglei Burkill ex Harms (Rimo Pagar), and C. x aurantiifolia (Rimo Bunga). Rimo Kejaren (C. × junos) is a species that has the most benefits.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.

Genome-wide analysis of NPR1-like genes in citrus species and expression analysis in response to citrus canker (Xanthomonas axonopodis pv. citri).

Citrus fruits, revered for their nutritional value, face significant threats from diseases like citrus canker, particularly impacting global citrus cultivation, notably in Pakistan. This study delves into the critical role of NPR1-like genes, the true receptors for salicylic acid (SA), in the defense mechanisms of citrus against Xanthomonas axonopodis pv. citri (Xcc). By conducting a comprehensive genome-wide analysis and phylogenetic study, the evolutionary dynamics of Citrus limon genes across diverse citrus cultivars are elucidated. Structural predictions unveil conserved domains, such as the BTB domain and ankyrin repeat domains, crucial for the defense mechanism. Motif analysis reveals essential conserved patterns, while cis-regulatory elements indicate their involvement in transcription, growth, response to phytohormones, and stress. The predominantly cytoplasmic and nuclear localization of NPR1-like genes underscores their pivotal role in conferring resistance to various citrus species. Analysis of the Ks/Ka ratio indicates a purifying selection of NPR1-like genes, emphasizing their importance in different species. Synteny and chromosomal mapping provide insights into duplication events and orthologous links among citrus species. Notably, Xac infection stimulates the expression of NPR1-like genes, revealing their responsiveness to pathogenic challenges. Interestingly, qRT-PCR profiling post-Xac infection reveals cultivar-specific alterations in expression within susceptible and resistant citrus varieties. Beyond genetic factors, physiological parameters like peroxidase, total soluble protein, and secondary metabolites respond to SA-dependent PR genes, influencing plant characteristics. Examining the impact of defense genes (NPR1) and plant characteristics on disease resistance in citrus, this study marks the inaugural investigation into the correlation between NPR1-associated genes and various plant traits in both susceptible and resistant citrus varieties to citrus bacterial canker.

DRIS standards for identifying yield-limiting nutrients in Khasi Mandarin and their relationship with fruit yield on acidic soil of the eastern Himalayas, India

Mandarin fruits are the most widely grown citrus fruits in the world, particularly in the eastern Himalayas of India, which is the primary center of citrus diversity. Diagnosing potential nutritional imbalances using leaf tissue analysis is a mandatory practice for a higher yield. Regarding the nutritional diagnosis of Khasi mandarin plants, particularly in the acidic soil of the hilly ecology of the Eastern Himalayas, India, there is currently no information available. Therefore, the present study was conducted to identify nutrient constraints, establish leaf-based diagnostic recommended and integrated system (DRIS) norms, and determine nutrients’ relationship with fruit yields in 144 orchards. The result showed that the DRIS indices predicted a nutritional optimum value for Khasi mandarin: 2.26–2.83% N, 0.11–0.16% P, 1.86–2.07% K, 1.85–2.12% Ca, 0.33–0.48% Mg, 170.10–225.10 mg kg−1 Fe, 74.03–83.43 mg kg−1 Mn, 1.24–2.45 mg kg−1 Cu, and 19.84–21.28 mg kg−1 Zn. The DRIS norms identified the nutrients Zn, P, Ca, K, N, and Mg as deficient to low levels (14.15→2.14), while Fe, Mn, and Cu were at high to excessive levels (5.50→18.25). The nutritional balance index had a significantly negative relationship with the fruit yield. Leaf nutrient concentrations of N (0.909**), P (0.827**), K (0.867**), Zn (0.833**), Ca (0.827**), and Mg (0.592**) had a significant positive correlation with fruit yield. The finding will facilitate the correct interpretation of leaf nutrient analysis, and the norms developed will enable a precise intervention through nutrient management for higher yields in mandarin citrus.

TINJAUAN PUSTAKA SISTEMATIS: KARAKTERISASI KEANEKARAGAMAN GENETIK SPESIES JERUK (Citrus sp.) MENGGUNAKAN PENANDA SIMPLE SEQUENCE REPEAT (SSR)

Background: The biodiversity of citrus plants is very abundant and many are cultivated, creating various types. However, the high demand makes orange farmers feel overwhelmed. This is what causes some orange cultivars to become rare. Characterization of the genetic diversity of citrus germplasm at the molecular level is an important and crucial step that can assist in the breeding and conservation process. The aim of this literature review is to provide information regarding molecular markers regarding the application of SSR molecular markers to analyze the genetic diversity of citrus. Method: The method used is a systematic literature review with the literature sources used coming from articles taken from national journals and international journals indexed by Scopus. Results: The application of molecular markers using the SSR method is useful for identifying and characterizing the genetic diversity of citrus germplasm so that it can assist in breeding and conservation actions. SSR has many advantages both in terms of its use and the results obtained. Conclusion: One application of molecular markers is to analyze plant genetic diversity. Simple Sequence Repeat (SSR) molecular markers can help to analyze the characteristics and genetic diversity of oranges so that they can assist in plant breeding and conservation purposes.

Design of smart citrus picking model based on Mask RCNN and adaptive threshold segmentation.

Smart agriculture is steadily progressing towards automation and heightened efficacy. The rapid ascent of deep learning technology provides a robust foundation for this trajectory. Leveraging computer vision and the depths of deep learning techniques enables real-time monitoring and management within agriculture, facilitating swift detection of plant growth and autonomous assessment of ripeness. In response to the demands of smart agriculture, this exposition delves into automated citrus harvesting, presenting an ATT-MRCNN target detection model that seamlessly integrates channel attention and spatial attention mechanisms for discerning and identifying citrus images. This framework commences by subjecting diverse citrus image classifications to Mask Region-based CNN's (Mask RCNN's) discerning scrutiny, enhancing the model's efficacy through the incorporation of attention mechanisms. During the model's training phase, transfer learning is utilized to expand data performance and optimize training efficiency, culminating in parameter initialization. Empirical results notably demonstrate that this method achieves a recognition rate surpassing the 95% threshold across the three sensory recognition tasks. This provides invaluable algorithmic support and essential guidance for the imminent era of intelligent harvesting.

Quantifying the ultraviolet-induced fluorescence intensity in green mould lesions of diverse citrus varieties: Towards automated detection of citrus decay in postharvest

Citrus fungal infections developing during fruit storage and transportation can cause significant economic losses after harvest. The most important is caused by the fungus Penicillium digitatum, which infects the fruit through rind wounds and causes a rot lesion. The symptoms of decay are difficult to notice by the human eye in the initial stages of decay development because the colour of the lesion is very similar to that of the healthy rind. One method to detect this disease early is to illuminate the fruit with ultraviolet (UV) light since the disease causes visible fluorescence. Manual inspection exposes the workers to UV light, which is dangerous for their skin and eyes. An alternative is to use artificial vision systems. But not all varieties show the same level of fluorescence, and even some do not produce this phenomenon, making it challenging to create effective automatic detection systems based on image analysis. This work has studied and determined the fluorescence level of 104 varieties of oranges and mandarins using hyperspectral and colour imaging. The samples were inoculated with spores of the P. digitatum in controlled conditions. Images were captured exposing the fruit under UV light (380 nm) using a colour camera and a hyperspectral imaging system. The fluorescence level of each variety was measured using three colour coordinates and the hyperspectral images. Best correlations between the spectral and the colour-based systems were achieved using the green (G) colour coordinate of the RGB colour space (R2 =0.85). Navel and common oranges emit the most fluorescence, while 16 varieties (mostly blood oranges and other mandarins) have very low or undetectable fluorescence.

Plant growth-promoting traits of culturable seed microbiome of citrus species from Purvanchal Himalaya.

Despite Northeastern India being "Treasure House of Citrus Genetic Wealth," genetic erosion of citrus diversity poses severe concern with a corresponding loss in seed microbial diversity. The seed microbiome of citrus species unique to the Purvanchal Himalaya is seldom explored for their use in sustainable orchard management. Isolation and characterization of culturable seed microbiomes of eight citrus species, namely, Citrus reticulata Blanco, C. grandis (L.) Osbeck, C. latipes Tanaka, C. megaloxycarpa Lushaigton, C. jambhiri Lush, C. sinensis (L.) Osbeck, C. macroptera Montr, and C. indica Tanaka collected from NE India were carried out. The isolates were then screened for an array of plant growth-promoting (PGP) traits [indole acetic acid (IAA) production, N2 fixation, phosphate and zinc complex dissolution, siderophores, and Hydrogen Cyanide (HCN) production]. The pure culture isolates of seed microbiomes were capable of dissolving insoluble Ca3(PO4)2 (1.31-4.84 µg Pi ml-1 h-1), Zn3(PO4)2 (2.44-3.16 µg Pi ml-1 h-1), AlPO4 (1.74-3.61 µg Pi ml-1 h-1), and FePO4 (1.54-4.61µg Pi ml-1 h-1), mineralized phytate (12.17-18.00 µg Pi ml-1 h-1) and produced IAA-like substances (4.8-187.29 µg ml-1 h-1). A few isolates of the seed microbiome were also able to fix nitrogen, secrete siderophore-like compounds and HCN, and dissolve ZnSO4 and ZnO. The 16S ribosomal Ribonucleic Acid (rRNA)-based taxonomic findings revealed that Bacillus was the most dominant genus among the isolates across citrus species. Isolates CG2-1, CME6-1, CME6-4, CME6-5, CME6-9, CJ7-1, CMA10-1, CI11-3, and CI11-4 were identified as promising bioinoculants for development of microbial consortium having multifaceted PGP traits for nutritional benefits of nitrogen, phosphorus and zinc, and IAA hormonal benefits to citrus crops for better fitness in acid soils.

Genetic diversity of the genus Citrus in Tomini Bay coastal areas, Indonesia based on morphological characters

Abstract. Kandowangko NY, Febriyanti. 2023. Genetic diversity of the genus Citrus in Tomini Bay coastal areas, Indonesia based on morphological characters. BIodiversitas 24: 2938-2952. Citrus plants are one of the fruit plants that are widely consumed by the community, both for food, health, and customs. The existence of Citrus plants in the coastal area of Tomini Bay only grows wild and has not been cultivated intensively. Lack of information on the diversity of citrus and the use of Citrus plants that are not balanced with cultivation actions will threaten the existence of these Citrus plants. This research aims to provide information about the morphological characteristics of the genus Citrus in the coastal area of Tomini Bay. The plant materials used were 39 accessions consisting of 7 species. Descriptive methods and diversity index calculations as well as analysis of phenetic consanguinity data were applied in this study. The results showed that all Citrus accessions have morphological diversity ranging from stems, leaves, flowers, fruits, and seeds that are influenced by genetic and environmental factors. The diversity index (H’) was equal to 0.8103 which was categorized as low. The 39 Citrus accessions were divided into two main clusters with a similarity index of 64.3%. The findings of this study are expected to be useful in Citrus breeding and conservation programs in the future.

Characterization of miniature inverted repeat transposable elements inserted in the CitRWP gene controlling nucellar embryony and development of molecular markers for reliable genotyping of CitRWP in Citrus species

Nucellar embryony, a form of apomixis, is one of the most important traits in citrus breeding. Transposition of a miniature inverted repeat transposable element (MITE) in the promoter of CitRWP gene has been previously reported as the causal mutation. However, features of the MITE such as sequences of terminal inverted repeats (TIRs) and target site duplications (TSDs) remain unclear. An MITE family, DTM10, showing the highest homology was identified from 110 citrus MITE families. At least 80 DTM10 elements were identified from each of 10 genome sequences of Citrinae species. Analysis of 114 sweet orange elements showed that DTM10 contained 56-bp TIRs and mostly 9-bp TSDs. Phylogenetic analyses of DTM10 elements identified from five Citrinae species showed that MITEs in CitRWP might have transposed in a common ancestor of Citrus species. Two inverted MITEs were identified from three mutant alleles analyzed. In addition, three tandem MITEs were identified in the mutant allele of Fortunella CitRWP. A strong DNA secondary structure formed between MITEs was predicted. Due to a strong stem-loop structure, a phenomenon of polymerase jumping was observed during PCR amplification. To develop reliable molecular markers, sequences of mutant and wild-type CitRWP alleles were obtained from diverse citrus accessions. Phylogenetic analysis of CitRWP alleles showed that mutant alleles might have originated from a common ancestor of Citrus species. Based on MITE insertion and a single nucleotide polymorphism in the genic region, three complementary molecular markers were developed. Genotyping results of 241 citrus accessions were found to be identical among three molecular markers.

Conserving Citrus Diversity: From Vavilov’s Early Explorations to Genebanks around the World

Citrus is among the most economically important fruit crops. Its vast species diversity and global production was observed by N.I. Vavilov during his international plant explorations from the early to mid-1900s. Currently, ex situ citrus collections located around the world conserve and protect citrus genetic resources, as revealed in a survey conducted in 2021. Responses were received from 43 collections in 27 countries, of which 35 provided data regarding collection composition, management practices, and security, as well as other information. The six largest citrus collections have between 1000 and 1735 accessions. The largest accession holdings are mandarins and sweet oranges, although all citrus fruit types are maintained: mandarin, sweet orange, lemon, pummelo, grapefruit, hybrids, lime, sour orange, citron, kumquat, papeda, finger lime, and crop wild relatives. Diseases pose significant threats to collections, though some collections are maintained in a clean-plant state as a result of intensive sanitation efforts. National and regional quarantine regulations often limit the export and import of citrus plants or propagative materials, thus limiting the availability of materials at an international level. Resources, both financial and human, are necessary to ensure the long-term safety and security of citrus collections on a global scale. Future efforts to develop citrus genebanking communities will provide opportunities for improved conservation, as well as collaborations and training.

Antimicrobial high molecular weight pectin polysaccharides production from diverse citrus peels using a novel PL10 family pectate lyase

The discovery of environmentally friendly enzymes that can convert inexpensive and abundant citrus peel pectin into high value-added product is a potential avenue for the citrus peel application. In this study, a novel PL10-family pectate lyase (pelA) was characterized from marine bacterium Echinicola pacifica. PelA was a Ca2+ dependent pectate lyase whose activity was highest at pH 8 and 40 °C. It was capable of degrading polygalacturonic acid (PGA) and citrus peel pectin (CPP), but not apple peel pectin. Notably, PelA hydrolyzed PGA to high molecular weight polysaccharide (average molecular weight 111.4 kDa). Moreover, PelA was also able to degrade CPP from nine distinct citrus species into polysaccharides (average molecular weight ranging from 84.7 to 539.2 kDa) that showed antimicrobial activity against Staphylococcus epidermidis (88.8 %), Bacillus subtilis (99.8 %), Staphylococcus aureus (92.1 %), Escherichia coli (100.0 %) and Klebsiella pneumoniae (86.4 %). Considering the high market value of pectin in the food industry, PelA's capacity to convert citrus pectin into high molecular weight polysaccharides lays a foundation for its applications.

Phytochemical content and antioxidant activity in eight citrus cultivars grown in Jeju Island according to harvest time

ABSTRACT This study aimed to compare phytochemical content and antioxidant activities of eight diverse citrus cultivars grown in Jeju Island depending on their harvest time owing to their health benefits. Plant peels, flesh, and leaves were extracted, and their flavonoid contents were measured by HPLC. Flavonoid levels were high in peel followed by leaves and juices. It was highest in immature fruits and rapidly declined during ripening. While total phenolic content in all citrus peels and fleshes reduced during ripening, those of leaves did not significantly decrease. Antioxidant activity matched the total phenolic content. This indicates that immature citrus tissues exhibited the highest flavonoid and phenolic content with high antioxidant activity and can be potentially employed as a readily accessible natural antioxidant source.

A versatile Agrobacterium-based plant transformation system for genetic engineering of diverse citrus cultivars

Developing an efficient transformation system is vital in genetically engineering recalcitrant crops, particularly trees. Here, we outline an Agrobacterium tumefaciens-based stable plant transformation methodology for citrus genetic engineering. The process was optimized to suit the requirements of fourteen citrus varieties by establishing appropriate infection, co-cultivation, selection, and culture media conditions. The procedure includes transforming seedling-derived epicotyl segments with an A. tumefaciens strain, then selecting and regenerating transformed tissues. Transgenic shoots were further identified by a visual reporter (e.g., β-glucuronidase) and confirmed by Northern and Southern blot analysis. Transgene integrations among the transgenic lines ranged between one to four. The methodology can yield transformation efficiencies of up to 11%, and transgenic plants can be recovered as early as six months, depending on the variety. In addition, we show that incorporating A. tumefaciens helper virulence genes (virG and virE), spermidine, and lipoic acid in the resuspension buffer before transformation improved the transformation efficiency of specific recalcitrant cultivars, presumably by enhancing T-DNA integration and alleviating oxidative stress on the explant tissues. In conclusion, the optimized methodology can be utilized to engineer diverse recalcitrant citrus varieties towards trait improvement or functional genetics applications.

Analysis of ‘Marrakesh limetta’ (Citrus × limon var. limetta (Risso) Ollitrault, Curk & R.Krueger) horticultural history and relationships with limes and lemons

Citrus (Rutaceae) species have a relevant role in the culture of the Mediterranean countries, not only for their gastronomic or medicinal value, but especially for their ornamental and symbolic importance. The interest on Citrus diversity led to collect and cultivate numerous cultivars around the Mediterranean Sea, at least since the Roman period. Their image is documented in mosaics, paintings, sculptures and coins since the first millennium BC. Among these, sweet and sour limettas Citrus × limon var. limetta (Risso) Ollitrault, Curk & R.Krueger stand for their history and conservation status. Presently sour limetta is cultivated in Morocco, especially in Marrakesh, and in the Valle de Ricote (Murcia, Spain). Our work has three main objectives: to characterize sour limetta on the basis of Spanish and Moroccan populations, as well as its cultural relevance throughout history, especially in painting, gastronomy, agriculture and gardening, to determine their relationships with limes and lemons and to evaluate the availability and state of conservation of that legacy.We analyze and illustrate the morphology of the plant, flowers and fruits of sour limetta and their differences with other related citrus species and cultivars: sweet limetta, lemon, common lime, Persian lime, bergamot, mellarosa, Meyer lemon, rough lemon, Pursha Romana lime, Palestine sweet lime, Pomum Adami lemon and Rangpur lime. The above species and cultivars were selected on the base of their known ancestry and pomological characters. The distinctive characters are useful for the identification of sour limetta in the field but also for the identifying images of this plant in paintings. We analyzed the ancestry of the different taxa involved and compared the classification based on phenotypes with the classification based on the proportion of ancestors’ genomes in each individual taxa. The image of sour limetta appears in different paintings, from the van Eyck's Ghent Polyptych (early 15th century), and numerous still life works by Italian and Spanish authors of the 16th to 18th centuries. A peculiar type of evidence is provided by presence of limetta in the pictorial catalogues of the Medici's fruit collections displayed in a series of paintings by Bartolomeo Bimbi.This distinctly sour limetta is sold in the markets of Rabat (and other cities of Morocco), either fresh, candied or brined and canned. It is an important ingredient of traditional Moroccan cuisine. Although sour lime was a widely used ingredient in Italian cuisine during the 17th century, its state of preservation is deplorable outside of Morocco, and even there it is gradually replaced by lemons. We recommend its cultivation as a resource both for obtaining rootstocks as well as for its fruits, both for Moroccan cuisine and that of other parts of the world for the excellent aromas and flavors that they contribute to the dishes. In this way, by promoting its crop, we will prevent it from disappearing. It is also important to preserve sour lime in germplasm banks.

RAPD primer screening as a preliminary study to analyze the genetic diversity of Citrus spp. in South Sulawesi, Indonesia

The typical citrus germplasm collection in South Sulawesi has not been thoroughly characterized, especially in several citrus development centers, which have begun to be promoted again after the decline in productivity due to CVPD infection. The study of citrus diversity is very important to support future citrus breeding programs. Random amplified polymorphic DNA (RAPD) has been widely used for the analysis of genetic diversity among species in populations. In this study, 23 RAPD primers were used on Citrus cultivated in Selayar and Pangkep Regencies, which are citrus development areas in South Sulawesi. A total of 19 primers (OPA-05, OPA-09, OPA-17, OPC-09, OPC-17, OPE-04, OPH-04, OPH-15, OPN-14, 0PN-16, OPR-08, OPR-20, OPW-06, OPW-09, OPX-07, OPX-11, OPX-17, UBC-18, and UBC-51) can form polymorphic bands in randomly selected DNA samples. Monomorphic bands were formed by OPA-12 and OPD-07 primer in 12 samples. The primers OPX-13 and OPX-16 produced unclear bands. These 19 primers can be used to amplify DNA and determine the genetic diversity of Citrus in further analysis.

Multiomics-based dissection of citrus flavonoid metabolism using a Citrus reticulata \xd7 Poncirus trifoliata population

Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes. Although citrus plants are among the most important fruit crops worldwide, the genetic basis of secondary metabolism in these plants is largely unknown. Here, we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues (young leaf, old leaf, mature pericarp, and mature pulp) of an F1 pseudo-testcross citrus population. We detected 80 flavonoids in this population and identified 138 quantitative trait loci (QTLs) for 57 flavonoids in these four tissues. Based on transcriptional profiling and functional annotation, twenty-one candidate genes were identified, and one gene encoding flavanone 3-hydroxylase (F3H) was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions. The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.