Citrus Cultivars Research Articles

Genomic analysis provides insights into the origin and divergence of fruit flavor and flesh color of pummelo.

Pummelo (Citrus maxima) is one of the most important citrus crops and have genetically contributed sweet orange, lemon and most citrus cultivars. It has been cultivated for c. 4000 years in China and is also distributed in many Southeast Asian countries. Nevertheless, the origin and dispersal of pummelo remain elusive. We conducted whole-genome sequencing for 290 pummelo accessions from China and Southeast Asia (SEA). Our findings indicated that pummelo was originated in Yunnan province. The divergence of the China-SEA accessions occurred c. 2000 years ago and the divergence was likely facilitated through the Maritime Silk Road. We detected the divergence of genomic regions associated with fruit flavor and color, indicating different selection by human activities in different regions. A gene encoding lycopene cyclase 2 (LCYB2) exhibited a high degree of divergence in expression and sequence between red-flesh and white-flesh pummelos. A SNP in the coding region of LCYB2 resulted in a reduction in lycopene β-cyclizing enzyme activity, leading to the accumulation of lycopene and the development of the red-flesh trait. This study reveals the origin and evolutionary history of pummelo and provides insights into the genomic basis for the divergence of fruit flavor and color.

Citrus PR4A is involved in the defense responses against Xanthomonas citri subsp. citri.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a globally quarantine disease that infect nearly all Citrus cultivars. Remarkably, Citron C-05 has been identified with complete and active resistance to Xcc. However, the mechanism underlying Citron C-05’s resistance to Xcc remains elusive. In this study, we identified a gene cluster on the chromosome 8 of citrus genome comprising five pathogenesis-related 4-like genes. PR4A was up-regulated in Citron C-05 leaves under Xcc infection, exhibiting the highest expression among these PR4-like genes. In addition, PR4A expression was higher in leaves of disease-resistant genotypes compared to susceptible genotypes under Xcc invasion. Bimolecular fluorescence complementation (BiFC) and Split-Luc assays indicated that CmWRKY75, a <pg=>positive regulator of PR4A, interacted with pthA4 and up-regulated expression of PR4A in Citron C-05 leaves. Segmentation of the CmPR4A promoter revealed that PCmPR4A-P-516 may be involved in regulating PR4A expression. Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange, and three transgenic lines of OE-CmPR4A exhibited resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis. Furthermore, CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait, BiFC and Split-Luc assays further verified their interaction. Transient overexpression of CmSMU2 in the sweet orange increased resistance to Xcc. Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A, indicating that they may synergistically enhance resistance to Xcc in citrus. These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.

Comparative Analysis of the β-Cryptoxanthin Content of Various Citrus Cultivars in Jeju, Korea

Comparative Analysis of the β-Cryptoxanthin Content of Various Citrus Cultivars in Jeju, Korea

Gene expression profiles and metabolic pathways responsible for male sterility in cybrid pummelo.

Abnormal expression of genes regulating anther and pollen development and insufficient accumulation of male sterility (MS)- related metabolites lead to MS in cybrid pummelo Male sterility (MS) is a major cause of seedlessness in citrus, which is an important trait for fresh fruit. Understanding the mechanism of MS is important for breeding seedless citrus cultivars. In this study, we dissected the transcriptional, metabolic and physiological mechanisms of MS in somatic cybrid of pummelo (G1 + HBP). G1 + HBP exhibited severe male sterility, manifesting as retarded anther differentiation, abnormal anther wall development (especially tapetum and endothecium), and deficient pollen wall formation. In the anthers of G1 + HBP, the expression of genes regulating anther differentiation and tapetum development was abnormal, and the expression of genes regulating endothecium secondary lignification thickening and pollen wall formation was down-regulated. The transcription of genes involved in MS-related biological processes, such as jasmonic acid (JA) signaling pathway, primary metabolism, flavonoid metabolism, and programmed cell death, was altered in G1 + HBP anthers, and the accumulation of MS-associated metabolites, including fatty acids, amino acids, sugars, ATP, flavonols and reactive oxygen species (ROS), was down-regulated in G1 + HBP anthers. In summary, abnormal expression of key genes regulating anther and pollen development, altered transcription of key genes involved in MS-related metabolic pathways, and insufficient accumulation of MS-related metabolites together lead to MS in G1 + HBP. The critical genes and the metabolism pathways identified herein provide new insights into the formation mechanism of MS in citrus and candidate genes for breeding seedless citrus.

Study of Growth Characteristics of Some Citrus Cultivars in the Juvenile Stage under Imereti Conditions

Study of Growth Characteristics of Some Citrus Cultivars in the Juvenile Stage under Imereti Conditions

Genomic and metabolomic insights into the selection and differentiation of bioactive compounds in citrus

Bioactive compounds are playing an increasingly prominent role in breeding functional and nutritive fruit crops such as citrus. However, the genomic and metabolic basis for the selection and differentiation underlying bioactive compounds variations in citrus remain poorly understood. Here, we constructed a species-level variation atlas of genomes and metabolomes using 299 citrus accessions. A total of 19,829 significant SNPs were targeted to 653 annotated metabolites, among which multiple significant signals were identified for secondary metabolites, especially flavonoids. Significantly differential accumulation of bioactive compounds in phenylpropane pathway, mainly flavonoids and coumarins, were unveiled across ancestral citrus species during differentiation, which is likely associated with the divergent haplotype distribution and/or expression profiles of relevant genes, including p-coumaroyl coenzyme A 2'-hydroxylases, flavone synthases, cytochrome P450 enzymes, prenyltransferases and UDP-glycosyltransferases. Moreover, we elucidated the citrus varieties with excellent antioxidant and anticancer capacities, clarifying the robust associations between distinct bioactivities and specific metabolites. Thus, these findings provide citrus breeding options for enrichment of beneficial flavonoids and avoidance of the potential risk of coumarins. This study will illuminate the application of genomic and metabolic engineering strategies in developing modern healthy citrus cultivars.

Microcapsule Preparation and Properties of Flavonoid Extract from Immature Citrus reticulata 'Chachiensis' Peel.

Citrus reticulata 'Chachiensis' is a citrus cultivar in the Rutaceae family, and its peel is commonly utilized as a raw material for Guangchenpi. This study used flavonoid extract from the peel of immature Citrus reticulata 'Chachiensis' (CCE) as the raw material to investigate the encapsulation ability of different wall materials (plant-based proteins, including soybean protein isolation (SPI), pea protein (PP), and zein; carbohydrates, including maltodextrin (MD), Momordica charantia polysaccharide (MCP), and gum acacia (GA); and composite wall materials of both types) on CCE. The wall material with the highest encapsulation rate was selected for the preparation of CCE microcapsules. Furthermore, the physicochemical characteristics, antioxidant capacity, bioavailability, and storage stability of the CCE microcapsules were explored. The results indicated that among all wall materials, the composite wall material PPMD had the highest encapsulation rate, which was 84.44 ± 0.34%. After encapsulation, the microcapsules tended to have a yellow color and exhibited characteristics such as system stability, low moisture content, and low hygroscopicity. In vitro antioxidant assays revealed that the encapsulation of CCE significantly increased the scavenging rates of DPPH and ABTS free radicals. In vitro gastrointestinal digestion experiments indicated that the release rate of PPMD-CCE in intestinal fluid was significantly greater than that of free CCE, ultimately reaching 85.89 ± 1.53%. Storage experiments demonstrated that after 45 days under various temperature and light conditions, the retention rate of CCE in the microcapsules was significantly greater than that of free CCE. The above findings provide new possibilities for the application of PP and plant proteins and lay a foundation for the future industrial application of CCE.

Animal pollination contributes to more than half of citrus production

Animal pollination is crucial for the reproduction and economic viability of a wide range of crops. Despite the existing data, the extent to which citrus crops depend on pollinators to guarantee fruit production still needs to be determined. Here, we described the composition of potential pollinators in citrus (Citrus spp.) from the main growing areas of Argentina; moreover, we combined Bayesian models and empirical simulations to assess the contribution of animal pollination on fruit set and yield ha−1 in different species and cultivars of lemons, grapefruits, mandarins, and oranges. Honeybee (A. mellifera L.) was the most commonly observed potential pollinator, followed by a diverse group of insects, mainly native bees. Regardless of citrus species and cultivars, the probability of flowers setting fruit in pollinated flowers was 2.4 times higher than unpollinated flowers. Furthermore, our simulations showed that about 60% of the citrus yield ha−1 can be attributable to animal pollination across all species and cultivars. Therefore, it is crucial to maintain environments that support pollinator diversity and increase consumer and to producer awareness and demand in order to ensure the significant benefits of animal pollination in citrus production.

The Ectopic Expression of the MpDIR1(t) Gene Enhances the Response of Plants from Arabidopsis thaliana to Biotic Stress by Regulating the Defense Genes and Antioxidant Flavonoids.

The Defective in Induced Resistance 1 (DIR1) gene, a member of the lipid transferase proteins (LTPs), plays a crucial role in plant defense against pathogens. While previous transcriptomic studies have highlighted the significant expression of citrus LTPs during biotic stress, functional annotations of LTPs in the Citrus genera remain limited. In this study, we cloned the Murraya paniculata DIR1 (MpDIR1(t)) gene and overexpressed it in Arabidopsis thaliana to evaluate its stress response mechanisms against biotic stress. The transgenic Arabidopsis lines showed fewer disease symptoms in response to Pseudomonas syringae (Pst DC3000) compared to wild-type Arabidopsis. Defense and pathogenesis-responsive genes such as PR1, PR4, PR5, and WRKY12 were significantly induced, showing a 2- to 12-fold increase in all transgenic lines compared to the wild type. In addition, the Pst DC3000-infected transgenic Arabidopsis lines demonstrated elevated levels of flavonoids and salicylic acid (SA), along with higher expression of SA-related genes, compared to the wild type. Moreover, all transgenic lines possessed lower reactive oxygen species levels and higher activity of antioxidant defense enzymes such as superoxide dismutase, peroxidase, and catalase under Pst DC3000 stress compared to the wild type. The up-regulation of defense genes, activation of the SA pathway, accumulation of flavonoids, and reinforcement of antioxidant defense mechanisms in transgenic Arabidopsis lines in response to Pst DC3000 underscore the critical role of MpDIR1(t) in fortifying plant immunity. Thus, MpDIR1(t) constitutes a promising candidate gene for improving bacterial disease resistance in commercial citrus cultivars.

CcCHIL, a type IV chalcone isomerase that can improve (2S)-naringenin production in Saccharomyces cerevisiae

(2S)-Naringenin, a crucial precursor in plant flavonoid synthesis, holds significance in diverse biological processes and potential therapeutic applications for human diseases. The economically valuable Citrus reticulata 'Chachi' citrus cultivar, renowned for its flavonoid-rich peel, was subjected to integrated transcriptomic and metabolomic analysis in this study to reveal patterns of flavonoid accumulation. The analysis revealed a strong correlation between the expression pattern of the gene encoding type IV chalcone isomerase (chalcone isomerase like, CHIL), CcCHIL, and the accumulation of flavonoids in C. reticulata 'Chachi' peel. Previous studies have demonstrated that type IV chalcone isomerase can enhance flavonoid accumulation in citrus and improve the catalytic efficiency of chalcone synthase in vitro, thereby increasing the titer of (2S)-naringenin. In this study, we constructed a genetically engineered yeast strain capable of de novo synthesis of (2S)-naringenin. We found that CcCHIL can increase (2S)-naringenin production in engineered yeast by 13.60 %. Subsequently, we conducted experiments using CHILs from various species and found that GmCHIL from Glycine max can increase (2S)-naringenin production in engineered yeast by 45.35 %. Sequence alignment, molecular docking predictions and site-directed mutagenesis showed that Asn151 is one of the key sites for CHIL improving (2S)-naringenin production. Our study unveiled CcCHIL's crucial role in flavonoid biosynthesis in C. reticulata 'Chachi' peel and paving the way for future synthesis of intricate and economically valuable flavonoids.

Whole genome sequences of 135 “Candidatus Liberibacter asiaticus” strains from China

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-limited alpha-proteobacteria causing Citrus Huanglongbing, the destructive disease currently threatening global citrus industry. Genomic analyses of CLas provide insights into its evolution and biology. Here, we sequenced and assembled whole genomes of 135 CLas strains originally from 20 citrus cultivars collected at ten citrus-growing provinces in China. The resulting dataset comprised 135 CLas genomes ranging from 1,221,309 bp to 1,308,521 bp, with an average coverage of 675X. Prophage typing showed that 44 strains contained Type 1 prophage, 89 strains contained Type 2 prophage, 44 strains contained Type 3 prophage, and 34 of them contained more than one type of prophage/phage. The SNP calling identified a total of 5,090 SNPs. Genome-based phylogenetic analysis revealed two major clades among CLas strains, with Clade I dominated by CLas strains containing Type 1 prophage (79/95) and Clade II dominated by CLas strains containing Type 1 or Type 3 prophage (80/95). This CLas genome dataset provides valuable resources for studying genetic diversity and evolutionary pattern of CLas strains.

The Discovery of a Citrus Yellow Vein Clearing Virus Hacienda Heights Isolate Diversifies the Geological Origins of the Virus in California, United States.

The citrus yellow vein clearing virus (CYVCV) is an emerging threat to the U.S. citrus industry. Reports from China shows it cause significant reductions in fruit yield and growth, particularly in lemon trees. In 2022, CYVCV was detected in a wide range of citrus cultivars in localized urban properties in Tulare, California. In 2024, a CYVCV-infected lemon tree was detected in Hacienda Heights in Los Angeles County, California, geographically separated from the Tulare foci. Through long-read sequencing technology, the whole-genome sequence of a CYVCV isolate from Hacienda Heights (designated as CYVCV-CA-HH1, Accession number PP840891.1) was obtained. Sequence alignments and neighbornet analysis strongly suggested that the CYVCV-CA-HH1 isolate has a different origin than the Tulare CYVCV (CYVCV CA-TL) isolates. The CYVCV CA-TL isolates were grouped with those from South Asia (India and Pakistan) and the Middle East (Türkiye), while the CYVCV-CA-HH1 isolate was grouped with isolates from East Asia (China and South Korea). Maximum likelihood phylogenetic analysis further supports this finding, showing that the CYVCV-CA-HH1 isolate shares the most recent common ancestor with a South Korean lineage, which derives from Chinese isolates. Together, our data suggest a diverse geological origin of CYVCV isolates in California.

Genetic Characteristics and Volatile Organic Compounds of Leaves and Fruit Edible Parts of Citrus depressa Hayata from Different Geographic Origins

Citrus depressa Hayata is a citrus cultivar grown in Japan and Taiwan. To assess the differences in genetic characteristics and volatile organic components (VOCs) in the leaves and edible parts of the fruits of 23 C. depressa accessions from different geographic origins, the tissues were analyzed using cleaved amplified polymorphic sequence markers and gas chromatography-mass spectrometry. A phylogenetic cluster analysis demonstrated that Kagoshima accessions had a close genetic relationship with one another, with Okinawan “Izumi kugani-like” being the most distinct accession. The predominant volatiles in the leaves were γ-terpinene, p-cymene, limonene, and linalool. Multivariate analysis and volcano plots revealed distinct volatiles in the leaves of each cultivation region: piperitone and citronellal (Kagoshima); 5,9,9-trimethyl-spiro[3.5]non-5-en-1-one (Okinawa); and hexanal (Taiwan). Furthermore, the edible parts of Taiwanese fruits contained abundant amounts of monoterpenes, including linalool and 1,8-cineole. In contrast, Kagoshima and Okinawa accessions were rich in aldehydes and esters, respectively. In conclusion, the genetic and volatile profiles of 23 C. depressa accessions of different origins could be distinguished, and multivariate analysis suggested that C. depressa contains diverse VOCs depending on where it is cultivated. These findings demonstrate the exclusivity of C. depressa resources in each region, which could assist farmers and agro-industries in promoting food products derived from C. depressa fruits.

Workflow for development of CAPS markers with one type of restriction enzyme to identify citrus cultivars

Given the ease of propagating fruit tree species through cloning, the economic viability of their breeding programs hinges on protecting breeders' rights. This necessitates the development of highly accurate DNA markers for cultivar identification. Here, we present a methodology for the rapid design of cleaved amplified polymorphic sequence (CAPS) markers to discriminate newly bred Japanese citrus cultivars from genetically related cultivars. We first compared the performance of ddRAD-seq and MIG-seq in citrus germplasm. The ddRAD-seq libraries generated using EcoRI and HindIII restriction enzymes yielded the highest number of polymorphisms. Subsequently, ddRAD-seq with EcoRI and HindIII was employed to analyze 29 citrus cultivars and thus identify 331,801 genome-wide polymorphisms. A semi-automated bioinformatics pipeline was then utilized to identify candidate CAPS markers, resulting in the discovery of 14,072 potential markers. Of these candidates, 52 were chosen for validation based on their recognition by the PstI restriction enzyme. This evaluation resulted in the development of 11 highly discriminative CAPS markers. Remarkably, a combination of only six such markers was sufficient to differentiate newly bred cultivars from their genetically related parents. The single restriction enzyme employed for these markers facilitates straightforward multiplexing. Finally, a combination of one multiplex marker testing two loci and four singleplex markers was successfully selected that completely discriminated the cultivars other than the bud sports used in this study. The pipeline established here extends beyond citrus and has the potential to simplify marker development and cultivar protection in various plant species.

Genotyping by sequencing reveals the genetic diversity of citrus cultivars cultivated in AlUla

Genotyping by sequencing reveals the genetic diversity of citrus cultivars cultivated in AlUla

Identification of Biomarkers Associated with Phyllosticta citricarpa Tolerance.

Citrus black spot (CBS) is a fungal disease caused by Phyllosticta citricarpa Kiely, (McAlpine Van der Aa), with most cultivars being susceptible to infection. Currently, disease control is based on the application of protective fungicides, which is restricted due to resistance, health and environmental concerns. Although using natural products for disease management is gaining momentum, more advances are required. This study obtained the metabolic profiles of the essential oil and cuticular waxes of two citrus cultivars with a varying susceptibility to CBS infection using gas chromatography-mass spectrometry. A multivariate data analysis identified possible biomarker compounds that contributed to the difference in susceptibility between the two cultivars. Several identified biomarkers were tested in vitro for their antifungal properties against P. citricarpa. Two biomarkers, propanoic acid and linalool, were able to completely inhibit pathogen growth at 750 mg/L and 2000 mg/L, respectively.

Furanocoumarin profiles and inhibitory effects on cytochrome P450 activity of whole citrus fruit

Abstract Furanocoumarins in citrus fruit can cause adverse drug interactions, however, there are few reports on furanocoumarins and drug interactions in common edible citrus cultivars except grapefruit. A UPLC-based method for ten furanocoumarins in citrus fruit was established and impacts on cytochrome P450 activity were investigated. Furanocoumarins were more abundant (2–43 times) in the peel than in the pulp. The pulp of Xiyou, Hongyou and Navel orange exhibited P450 inhibition with IC50 values of 0.63, 0.67 and 1.02 mg/mL, respectively. The peel of most varieties except Huyou and Satsuma exhibited P450 inhibition. Xiyou, Huangjinyou, Baiyou, Hongyou, Ponkan and Navel orange had IC50 values of 0.33, 0.38, 0.76, 0.35, 0.43 and 0.37 mg/mL, respectively. These findings indicate that the consumption of the pulp of popular Chinese varieties of citrus fruit except for Xiyou, Hongyou and Navel orange has a low risk of drug interactions, and use of citrus products including peel may carry a significant risk of adverse drug interactions.

Yield and Heat Unit Requirements for Several Citrus Cultivars over Several Seasons in Egypt

Citrus cultivars have various temperature needs for development and output at different times from flowering to harvesting, making climate one of the numerous factors that affect citrus’ productivity and quality. In this study, the yield and heat unit requirements for several commercial citrus species over several seasons from 2010/2011 to 2021/2022 in Egypt were investigated. For this investigation, the time of flowering and the time of fruit harvesting were recorded. However, the required heat units from flowering to fruit harvesting were calculated based on daily records of air temperature, taking into account that all results below zero (negative results) are not used and all maximum air temperatures above 35.0 °C (≥35.1) are changed to 35.0 °C. In addition, the base air temperature of 13.0 °C was utilized for determining the required heat units. The results showed that in the experimental area, the overall mean of minimum air temperature, maximum air temperature, air relative humidity, and precipitation during the experimental periods had values of 15.2 °C, 28.70 °C, 59.3%, and 0.3 mm/day, respectively. Moreover, the lowest number of days required from flowering to fruit harvesting was observed to be 290.0 days for Fremont mandarin. Furthermore, the highest number of days required from flowering to fruit harvesting was observed to be 482 and 440 days, respectively, for Bearss Seedless lime and Valencia orange. Our study has highlighted a notable diversity among the investigated citrus cultivars, particularly highlighting specific cultivars that possess high yield. The cultivar that shows the greatest yield over the investigated seasons of the study was Valencia orange with 147.6 kg/tree. Moreover, the average values of the heat unit requirements for Washington Navel orange, Valencia orange, Murcott mandarin, Fremont mandarin, and Bearss Seedless lime were 3112.7, 3628.3, 3221.9, 3027.6, and 4398.4 °C day, respectively. This is the first report regarding the heat unit’s determination for several citrus cultivars grown in Egypt, and we expect this research will provide a new awareness in accepting and discovering novel locations where citrus cultivars can be positively developed in Egypt. It will also prove to be a source of basic information for the development of the citrus crop model.

Metabolic responses to boron toxicity in different citrus cultivars

Metabolic responses to boron toxicity in different citrus cultivars

Functional Analysis of RMA3 in Response to Xanthomonas citri subsp. citri Infection in Citron C-05 (Citrus medica)

Citrus bacterial canker disease, caused by Xanthomonas citri subsp. citri (Xcc), poses a significant global threat to the citrus industry. Lateral organ boundaries 1 (Lob1) is confirmed as a citrus susceptibility gene that induces pathogenesis by interaction with the PthA4 effector of Xcc. Citron C-05 (Citrus medica) is a Citrus genotype resistant to Xcc. However, there is little information available on the regulation of Lob1 in resistant genotypes, which is important for the breeding of citrus cultivars resistant to canker disease. This study aimed to identify upstream regulatory factors of Lob1 in Citron C-05 and to investigate its function in disease resistance. ‘Bingtang’ sweet orange (C. sinensis), a susceptible genotype, was utilized as the control. cDNA yeast libraries of Xcc-induced Citron C-05 and ‘Bingtang’ sweet orange were constructed. The capacities of ‘Bingtang’ and Citron C-05 were 1.896 × 107 and 2.154 × 107 CFU, respectively. The inserted fragments ranged from 500 to 2000 bp with a 100% recombination rate. The promoter of Lob1 was segmented into two pieces and the P1 fragment from both genotypes was used to construct a bait yeast (PAbAi-CsLob1-P1; PAbAi-CmLob1-P1). Through library screening with the bait yeast, upstream regulators interacting with the Lob1-P1 promoter were identified and then validated using Y1H and dual-luciferase tests. The expression analysis of the three transcript factors indicated that RMA3 was upregulated by inoculation with Xcc in the resistant Citron C-05, but not in the susceptible sweet orange. The overexpression of CsRMA3 in ‘Bingtang’ sweet orange led to reduced canker symptoms, with a significantly lower pathogen density in the leaves following Xcc inoculation. When CmRMA3 was silenced by virus-induced gene silencing (VIGS) in Citron C-05, typical canker symptoms appeared on the CmRMA3-silenced leaves at 15 days post-inoculation with Xcc. Further expression analyses revealed that the CmRMA3 transcription factor suppressed the expression of Lob1. These results suggest that RMA3 participates in the resistant reaction of Citron C-05 to Xcc infection, and such a response might be in relation to its suppression of the expression of the pathogenic gene Lob1.