CRISPR-based genome editing: Catching impossibles for citrus improvements

Citrus is globally one of the major fruit crops, occupying a place of prominence in international trade and tariff through horticultural commodities. Despite such distinction, citrus crop is confronted with a variety of biotic and abiotic stresses, thereby, sustaining production is always a daunting task. The genome size of citrus is rather small, ranging from 265 to 400 MB, probably an advantage for controlled trait specific editing. The evolution of next generation sequencing has facilitated the whole genome sequencing of as many 10 citrus species with 16 draft genome sequences, offering near future possibility to develop genome tailored citrus species or inducing the desired genetic transformation to address the issues chronically ailing commercial citrus cultivation in India, which is by no mean, a simple task to accomplish. Despite genetically intrinsic challenges involved in generating transgenics in perennial crop like citrus, several transgenics have been developed in namely, sweet orange, lemon, and grapefruit loaded with some useful traits. But, the public perception and the time taken to develop trangenics in citrus and less success ratio led the researchers adapt alternate ways. Of late, the thumping success of genome editing tools, especially Clustered. Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has provided a new molecular tailoring machine for citrus improvement. In several citrus species like sweet orange, pummelo, and grapefruit CRISPR-Cas9 system has resulted in value added multiple traits-based transgenics. However, the major drawback of the CRISPR/Cas9 system is the generation of significant off-target cleavage sites as a result of complexing of gRNA with mismatched complementary target DNA within the genome. The use of CRISPR as genome editing technology is anticipated to induce many desired traits in citrus in years to come with more commercial applications in field for changed canopy structure, root traits, regular bearing, extended fruit maturity, besides multiple disease resistance.

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Patterns of genetic structure and evidence of Egyptian Citrus rootstock based on informative SSR, LTR-IRAP and LTR-REMAP molecular markers

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00128-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Mohamed Abd S. El zayat ◽

Ahmed H. Hassan ◽

Elsayed Nishawy ◽

Mohammed Ali ◽

Mohamed Hamdy Amar

Keyword(s):

Genetic Structure ◽

Structure Analysis ◽

Sweet Orange ◽

Group Structure ◽

Draft Genome ◽

Species Level ◽

Sour Orange ◽

Group Level ◽

Citrus Species ◽

Citrus Rootstock

Abstract Background Releasing the draft genome of sweet orange provides useful information on genetic structure and molecular marker association with heritable breeding traits in citrus species and their structures. Last decades, microsatellite and retrotransposons are well known as a significant diverse component of the structural evolution. They represented the most potent elements for assessing sustainable utilization of the complicated classification in citrus breeding. Our study was performed to verify the structure analysis and the parentage genetic diversity among the Egyptian citrus rootstocks and the related species. Results Here, the performance of 26 SSR and 14 LTR-IRAP in addition to 20 LTR-REMAP markers have been used to conduct the discriminating power and the status of the genetic structure analysis among twenty specimens of citrus genotypes. As a result, the three markers approach exhibited a remarkable variation among the tested genotypes. Overall, the three markers have different discrimination power; the co-dominant SSR markers can differentiate within the group level only in addition to the species level of sour orange, while the dominant markers LTR-IRAP had the ability to discriminate among the group level in addition to species level and the origin of acids. Similarly, LTR-REMAP is suitable for classifying the group level and species level for mandarins as well the origin of Egyptian acids; probably due to it is integration of SSR and LTR-IRAP techniques. Structure and PCoA results of LTR-REMAP marker in strong support for the group structure of citrus species have been divided into four sets: acids, grapefruit/pummelo, mandarin/orange, and sour orange. Conclusion Our findings of the genetic structure analysis support the monophyletic nature of the citrus species; are able to provide unambiguous identification and disposition of true species and related hybrids like lemon, lime, citron, sour orange, grapefruit, mandarin, sweet orange, pummelo, and fortunella; and resulted in their placement in individual or overlap groups. The outcomes of these results will offer helpful and potential information for breeding programs and conservation approaches as a key stage toward identifying the interspecific admixture and the inferred structure origins of Egyptian citrus rootstock and acid cultivars.

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Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

10.1101/2021.10.20.465103 ◽

2021 ◽

Author(s):

Xiaoen Huang ◽

Nian Wang

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Sweet Orange ◽

Susceptibility Gene ◽

Transformation Rate ◽

Biallelic Mutation ◽

Important Species ◽

Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

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A Social-Aware Service Recommendation Approach for Mashup Creation

International Journal of Web Services Research ◽

10.4018/jwsr.2013010103 ◽

2013 ◽

Vol 10 (1) ◽

pp. 53-72 ◽

Cited By ~ 9

Author(s):

Jian Cao ◽

Wenxing Xu ◽

Liang Hu ◽

Jie Wang ◽

Minglu Li

Keyword(s):

Social Relationships ◽

State Of The Art ◽

Value Added ◽

The Internet ◽

Service Recommendation ◽

Data Set ◽

Factorization Algorithm ◽

Comprehensive Service ◽

Daunting Task ◽

User Centric

Mashup is a user-centric approach to create value-added new services by utilizing and recombining existing service components. However, as services become increasingly more spontaneous and prevalent on the Internet, finding suitable services from which to develop a mashup based on users’ explicit and implicit requirements remains a daunting task. Several approaches already exist for recommending specific services for users but they are limited to proposing only services with similar functionality. In order to recommend a set of suitable services for a general mashup based on users’ functional specifications, a novel social-aware service recommendation approach, where multi-dimensional social relationships among potential users, topics, mashups, and services are described by a coupled matrices model, is proposed in this paper. Accordingly, a factorization algorithm is designed to predict unobserved relationships, and we use a genetic algorithm to learn some specific parameters, and then construct a comprehensive service recommendation model. Experimental results for a realistic mashup data set indicate that the proposed approach outperforms other state-of-the-art methods.

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Volatile Compounds in Fruit Peels as Novel Biomarkers for the Identification of Four Citrus Species

Molecules ◽

10.3390/molecules24244550 ◽

2019 ◽

Vol 24 (24) ◽

pp. 4550

Author(s):

Haipeng Zhang ◽

Huan Wen ◽

Jiajing Chen ◽

Zhaoxin Peng ◽

Meiyan Shi ◽

...

Keyword(s):

Volatile Compounds ◽

Sweet Orange ◽

Citrus Fruit ◽

Principal Component ◽

Citrus Species ◽

Alternative Approach ◽

Novel Biomarkers ◽

Volatile Profiles ◽

Volatile Biomarkers

The aroma quality of citrus fruit is determined by volatile compounds, which bring about different notes to allow discrimination among different citrus species. However, the volatiles with various aromatic traits specific to different citrus species have not been identified. In this study, volatile profiles in the fruit peels of four citrus species collected from our previous studies were subjected to various analyses to mine volatile biomarkers. Principal component analysis results indicated that different citrus species could almost completely be separated. Thirty volatiles were identified as potential biomarkers in discriminating loose-skin mandarin, sweet orange, pomelo, and lemon, while 17 were identified as effective biomarkers in discriminating clementine mandarins from the other loose-skin mandarins and sweet oranges. Finally, 30 citrus germplasms were used to verify the classification based on β-elemene, valencene, nootkatone, and limettin as biomarkers. The accuracy values were 90.0%, 96.7%, 96.7%, and 100%, respectively. This research may provide a novel and effective alternative approach to identifying citrus genetic resources.

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First Report of Xanthomonas citri pv. citri-A Causing Asiatic Citrus Canker in Mayotte

Plant Disease ◽

10.1094/pdis-01-13-0128-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 989-989

Author(s):

J. Hoarau ◽

C. Boyer ◽

K. Vital ◽

T. Chesneau ◽

C. Vernière ◽

...

Keyword(s):

Indian Ocean ◽

Sweet Orange ◽

Citrus Canker ◽

Negative Control ◽

Sterile Water ◽

Citrus Species ◽

Indian Ocean Region ◽

Southwest Indian Ocean ◽

Mexican Lime ◽

Ocean Region

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit and leaf drop and twig dieback on susceptible/very susceptible cultivars. A chlorotic halo is typically visible around canker lesions on leaves and young fruit, but not on mature fruit and twigs. This quarantine organism can strongly impact both national and international citrus markets. Long distance dispersal is mainly through infected propagative material. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region (Comoros, Mauritius, Reunion, Rodrigues, and Seychelles islands), but was not yet reported in Mayotte (EPPO-PQR available at http://www.eppo.int ). In May 2012, typical canker-like symptoms were observed on sweet orange (Citrus sinensis) groves on Mtsamboro islet and soon after on the main island of Mayotte, mostly on sweet oranges, but also on Tahiti limes (C. latifolia) and mandarins (C. reticulata). Eighty-one Xanthomonas-like strains were isolated using KC semi-selective medium (4) from disease samples collected from both commercial groves and nurseries on different Citrus species located all over the island. Sixteen Xanthomonas-like isolates were tentatively identified as X. citri pv. citri based on a specific PCR assay with 4/7 primers (3). All strains but the negative control, sterile water, produced an amplicon of the expected size similar to X. citri pv. citri strain IAPAR 306 used as positive control. Multilocus sequence analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified three strains from Mayotte (LJ225-3, LJ228-1, and LJ229-11) as X. citri pv. citri (and not other xanthomonad pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (2) (including all strains from the Southwest Indian Ocean region). Eight strains were inoculated by a detached leaf assay (2) to Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58, sweet orange cv. Washington Navel, alemow SRA 779 (C. macrophylla), and tangor cv. Ortanique (C. reticulata × C. sinensis) and developed typical erumpent, callus-like tissue at wound sites for all Citrus species, fulfilling Koch's postulates. Xanthomonas-like yellow colonies were reisolated from symptoms produced by the eight strains inoculated on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the negative control (sterile water). No lesions developed on the negative control consisting of inoculations by 10 mM tris buffer (pH 7.2). Citrus canker-free nurseries and grove sanitation should be implemented for decreasing the prevalence of Asiatic canker in this island territory. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

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Current Status of Microalgae to Produce Biogas through Pretreatment of Lignocellulosic Waste in the Era of 21-Century

10.20944/preprints202008.0233.v1 ◽

2020 ◽

Author(s):

Ali Raza Ishaq ◽

Faiza Jabeen ◽

Maleeha Manzoor ◽

Tahira Younis ◽

Ayesha Noor ◽

...

Keyword(s):

Fossil Fuels ◽

Value Added ◽

Raw Material ◽

Current Status ◽

Fish Feed ◽

Potential Candidate ◽

Lignocellulosic Waste ◽

Major Drawback ◽

Lignocellulosic Feedstocks ◽

Value Added Products

Modern day civilization is dependent on energy generation by fossil fuels. But the major drawback of using fossil fuels is environmental pollution. Microalgae are potential candidate for production of various products of interest, such as proteins, mini food, pigments and triglycerides that can be converted into biofuels. Lignocellulosic feedstocks are the most abundantly available raw material of plants that can serve as a promising feedstock for cultivating bacteria, fungi, yeasts and microalgae to produce biofuels and other value-added products. Owing to the abundant availability of these low/no cost substrates, can be utilized as feedstocks for cultivating microalgae to generate biogas/biodiesel. Likewise, there is much room to exploit defatted algal biomass to be used as animal/fish feed and oil producing/accumulating genes knowledge in future to produce high and good quality biodiesel and biogas.

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Improvement of Disease Resistance in Livestock: Application of Immunogenomics and CRISPR/Cas9 Technology

Animals ◽

10.3390/ani10122236 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2236

Author(s):

Md. Aminul Islam ◽

Sharmin Aqter Rony ◽

Mohammad Bozlur Rahman ◽

Mehmet Ulas Cinar ◽

Julio Villena ◽

...

Keyword(s):

Disease Resistance ◽

Genome Editing ◽

Public Perception ◽

Genetic Resistance ◽

Potential Candidate ◽

Technological Advances ◽

Disease Occurrence ◽

Host Genetic ◽

Safe Food ◽

Genomic Locations

Disease occurrence adversely affects livestock production and animal welfare, and have an impact on both human health and public perception of food–animals production. Combined efforts from farmers, animal scientists, and veterinarians have been continuing to explore the effective disease control approaches for the production of safe animal-originated food. Implementing the immunogenomics, along with genome editing technology, has been considering as the key approach for safe food–animal production through the improvement of the host genetic resistance. Next-generation sequencing, as a cutting-edge technique, enables the production of high throughput transcriptomic and genomic profiles resulted from host-pathogen interactions. Immunogenomics combine the transcriptomic and genomic data that links to host resistance to disease, and predict the potential candidate genes and their genomic locations. Genome editing, which involves insertion, deletion, or modification of one or more genes in the DNA sequence, is advancing rapidly and may be poised to become a commercial reality faster than it has thought. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) [CRISPR/Cas9] system has recently emerged as a powerful tool for genome editing in agricultural food production including livestock disease management. CRISPR/Cas9 mediated insertion of NRAMP1 gene for producing tuberculosis resistant cattle, and deletion of CD163 gene for producing porcine reproductive and respiratory syndrome (PRRS) resistant pigs are two groundbreaking applications of genome editing in livestock. In this review, we have highlighted the technological advances of livestock immunogenomics and the principles and scopes of application of CRISPR/Cas9-mediated targeted genome editing in animal breeding for disease resistance.

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Citrus Responses to Xylella fastidiosa Infection

Plant Disease ◽

10.1094/pdis-10-11-0868-re ◽

2012 ◽

Vol 96 (9) ◽

pp. 1245-1249 ◽

Cited By ~ 18

Author(s):

A. L. Garcia ◽

S. C. Z. Torres ◽

M. Heredia ◽

S. A. Lopes

Keyword(s):

Sweet Orange ◽

Control Strategies ◽

Xylella Fastidiosa ◽

Symptom Expression ◽

Xylem Anatomy ◽

Citrus Species ◽

Mechanisms Of Resistance ◽

Xylem Structure ◽

Citrus Varieties ◽

Inoculum Pressure

The xylem-limited bacterium Xylella fastidosa causes the widely disseminated citrus variegated chlorosis disease (CVC). In Brazil, CVC has been known for more than 20 years and affects only sweet orange. Lime and mandarin have remained free of symptoms despite the high inoculum pressure. Knowing the mechanisms underlying this apparent resistance is important to devise new disease control strategies. The reaction of commercial sweet orange (‘Caipira’, ‘Natal’, ‘Pêra’, and ‘Valencia’), lime (Mexican and Persian), mandarin (‘Cleopatra’, ‘Cravo’, ‘Ponkan’, and ‘Sunki’), and the acid citrus species Rangpur to X. fastidiosa was compared in artificially inoculated seedlings, which were assessed for symptom expression, pathogen isolation, and the amount of apparently occluded xylem vessels in cross-sectioned leaf petioles. Only the sweet orange expressed typical CVC symptoms, varying from 53.8% in Valencia to 63.0% in Caipira. Average percentages of positive isolations (pi) and occluded vessels (ov) were higher in sweet orange (pi= 59.2 to 75.0; ov = 10.7 to 25.8) than mandarin (pi = 2.3 to 16.3; ov = 1.4 to 4.0), lime (pi = 0 to 5.4; ov = 0 to 2.1), or Rangpur (pi = 1,9; ov = 1.1). There were no obvious differences in xylem anatomy among all citrus varieties, suggesting that the mechanisms of resistance to CVC are not related to any physical variation in xylem structure.

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Isoenzymatic polymorphism in Citrus spp. and Poncirus trifoliata (L.) Raf. (Rutaceae)

Genetics and Molecular Biology ◽

10.1590/s1415-47572000000100030 ◽

2000 ◽

Vol 23 (1) ◽

pp. 163-168 ◽

Cited By ~ 12

Author(s):

Valdenice Moreira Novelli ◽

Marcos Antonio Machado ◽

Catalina Romero Lopes

Keyword(s):

Leucine Aminopeptidase ◽

Sweet Orange ◽

High Variability ◽

Poncirus Trifoliata ◽

Polymorphism Analysis ◽

Citrus Species ◽

Shikimate Dehydrogenase ◽

Phosphogluconate Dehydrogenase ◽

Enzymatic Systems ◽

Citrus Spp

Isoenzymatic polymorphism analysis was used to determine genetic variability among species and hybrids of Citrus spp. and one accession of Poncirus trifoliata (L.) Raf. Ten enzymatic systems aspartate aminotransferase (AAT), acid phosphatase (ACP), leucine aminopeptidase (LAP), 6-phosphogluconate dehydrogenase (6-PGD), isocitrate dehydrogenase (IDH), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), diaphorase (DIA), shikimate dehydrogenase (SKD) and peroxidase (PRX) were analyzed. Twenty loci and 48 alleles were identified. Sweet orange cultivars (C. sinensis (L). Osbeck) showed the highest polymorphism with the largest number of heterozygous loci, although the alleles of those loci were the same in all cultivars, with the exception of Westin and Lima graúda. Mandarins (C. reticulata Blanco) exhibited diverse patterns, whereas Poncirus trifoliata (L.) Raf. showed high variability with all Citrus species and hybrids. Exclusive phenotypes were observed in some enzymatic systems, and similar patterns were found among interspecific hybrids and their putative parents.

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Citrus Biotechnology: Current Innovations and Future Prospects

10.5772/intechopen.100258 ◽

2021 ◽

Author(s):

Ghulam Mustafa ◽

Muhammad Usman ◽

Faiz Ahmad Joyia ◽

Muhammad Sarwar Khan

Keyword(s):

Tissue Culture ◽

Genome Editing ◽

Abiotic Stresses ◽

Fungal Pathogens ◽

Citrus Fruit ◽

Future Prospects ◽

Citrus Species ◽

Fruit Crop ◽

Wide Range ◽

Biology Research

Citrus is a valuable fruit crop worldwide. It not only provides essential minerals and vitamins but is also of great commercial importance. Conventional research has contributed a lot to the improvement of this fruit plant. Numerous improved varieties have been developed through conventional breeding, mutational breeding, polyploidization and tissue culture yet pathogens continue to emerge at a consistent pace over a wide range of citrus species. Citriculture is vulnerable to various biotic and abiotic stresses which are quite difficult to be controlled through conventional research. Biotechnological intervention including transgenesis, genome editing, and OMICS offers several innovative options to resolve existing issues in this fruit crop. Genetic transformation has been established in many citrus species and transgenic plants have been developed having the ability to tolerate bacterial, viral, and fungal pathogens. Genome editing has also been worked out to develop disease-resistant plants. Likewise, advancement in OMICS has helped to improve citrus fruit through the knowledge of genomics, transcriptomics, proteomics, metabolomics, interactomics, and phenomics. This chapter highlights not only the milestones achieved through conventional research but also briefs about the achievements attained through advanced molecular biology research.

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