CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.

Discrimination of Genetically Very Close Accessions of Sweet Orange (Citrus sinensis L. Osbeck) by Laser-Induced Breakdown Spectroscopy (LIBS).

The correct recognition of sweet orange (Citrus sinensis L. Osbeck) variety accessions at the nursery stage of growth is a challenge for the productive sector as they do not show any difference in phenotype traits. Furthermore, there is no DNA marker able to distinguish orange accessions within a variety due to their narrow genetic trace. As different combinations of canopy and rootstock affect the uptake of elements from soil, each accession features a typical elemental concentration in the leaves. Thus, the main aim of this work was to analyze two sets of ten different accessions of very close genetic characters of three varieties of fresh citrus leaves at the nursery stage of growth by measuring the differences in elemental concentration by laser-induced breakdown spectroscopy (LIBS). The accessions were discriminated by both principal component analysis (PCA) and a classifier based on the combination of classification via regression (CVR) and partial least square regression (PLSR) models, which used the elemental concentrations measured by LIBS as input data. A correct classification of 95.1% and 80.96% was achieved, respectively, for set 1 and set 2. These results showed that LIBS is a valuable technique to discriminate among citrus accessions, which can be applied in the productive sector as an excellent cost-benefit tool in citrus breeding programs.

Wide-ranging transcriptomic analysis of Poncirus trifoliata, Citrus sunki, Citrus sinensis and contrasting hybrids reveals HLB tolerance mechanisms.

Huanglongbing (HLB), caused mainly by 'Candidatus Liberibacter asiaticus' (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.

Expression Quantitative Trait Loci (eQTL) mapping for callose synthases in intergeneric hybrids of Citrus challenged with the bacteria Candidatus Liberibacter asiaticus.

Citrus plants have been extremely affected by Huanglongbing (HLB) worldwide, causing economic losses. HLB disease causes disorders in citrus plants, leading to callose deposition in the phloem vessel sieve plates. Callose is synthesized by callose synthases, which are encoded by 12 genes (calS1- calS12)in Arabidopsis thaliana. We evaluated the expression of eight callose synthase genes from Citrus in hybrids between Citrus sunki and Poncirus trifoliata infected with HLB. The objective of this work was to identify possible tolerance loci combining the expression quantitative trait loci (eQTL) of different callose synthases and genetic Single-Nucleotide Polymorphism (SNP) maps of C. sunki and P. trifoliata. The expression data from all CscalS ranged widely among the hybrids. Furthermore, the data allowed the detection of 18 eQTL in the C. sunki map and 34 eQTL in the P. trifoliata map. In both maps, some eQTL for different CscalS were overlapped; thus, a single region could be associated with the regulation of more than one CscalS. The regions identified in this work can be interesting targets for future studies of Citrus breeding programs to manipulate callose synthesis during HLB infection.

Carotenoid biosynthesis and quality characteristics of new hybrids between tangor (Citrus reticulata x C. sinensis) cv. 'Murcott' and sweet orange (C. sinensis) cv. 'Pêra'.

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. 'Murcott' and sweet orange cv. 'Pêra'. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly ß-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250-426 µg/g dry weight [dw]) and ß-cryptoxanthin (81-125 µg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 µg/g dw) and ß-cryptoxanthin (5-22 µg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 µg/g dw versus 158-250 µg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content.

QTLs and eQTLs mapping related to citrandarins' resistance to citrus gummosis disease.

BACKGROUND: Phytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) causes severe damage to citrus crops worldwide. A population of citrandarins was created from the cross between the susceptible parent Citrus sunki Hort. Ex Tan. and the resistant parent Poncirus trifoliata (L.) Raf. cv. Rubidoux, both parents and two reference rootstocks (Rangpur lime and Swingle citrumelo) were grafted in a greenhouse on Rangpur lime. Inoculations were performed at 10 cm and 15 cm above the grafting region and the resulting lesions were evaluated by measuring the lesion length 60 days after inoculation. As control, non-inoculated plants of each genotype were used. In addition, we evaluated the expression of 19 candidate genes involved in citrus defense response 48 h after pathogen infection by quantitative Real-Time PCR (qPCR). We mapped genomic regions of Quantitative Trait Loci (QTLs) and Expression Quantitative Trait Loci (eQTLs) associated with resistance to P. parasitica in the linkage groups (LGs) of the previously constructed maps of C. sunki and P. trifoliata. RESULTS: We found disease severity differences among the generated hybrids, with lesion lengths varying from 1.15 to 11.13 mm. The heritability of the character was 65%. These results indicate that there is a great possibility of success in the selection of resistant hybrids within this experiment. The analysis of gene expression profile demonstrated a great variation of responses regarding the activation of plant defense pathways, indicating that citrandarins have several defense strategies to control oomycete infection. The information of the phenotypic and gene expression data made possible to detect genomic regions associated with resistance. Three QTLs and 84 eQTLs were detected in the linkage map of P. trifoliata, while one QTL and 110 eQTLs were detected in C. sunki. CONCLUSIONS: This is the first study to use eQTLs mapping in the Phytophthora-citrus interaction. Our results from the QTLs and eQTLs mapping allow us to conclude that the resistance of some citrandarins to the infection by P. parasitica is due to a favorable combination of QTLs and eQTLs transmitted by both parents.

QTL mapping for fruit quality in Citrus using DArTseq markers.

BACKGROUND: Citrus breeding programs have many limitations associated with the species biology and physiology, requiring the incorporation of new biotechnological tools to provide new breeding possibilities. Diversity Arrays Technology (DArT) markers, combined with next-generation sequencing, have wide applicability in the construction of high-resolution genetic maps and in quantitative trait locus (QTL) mapping. This study aimed to construct an integrated genetic map using full-sib progeny derived from Murcott tangor and Pera sweet orange and DArTseq™ molecular markers and to perform QTL mapping of twelve fruit quality traits. A controlled Murcott x Pera crossing was conducted at the Citrus Germplasm Repository at the Sylvio Moreira Citrus Centre of the Agronomic Institute (IAC) located in Cordeirópolis, SP, in 1997. In 2012, 278 F1 individuals out of a family of 312 confirmed hybrid individuals were analyzed for fruit traits and genotyped using the DArTseq markers. Using OneMap software to obtain the integrated genetic map, we considered only the DArT loci that showed no segregation deviation. The likelihood ratio and the genomic information from the available Citrus sinensis L. Osbeck genome were used to determine the linkage groups (LGs). RESULTS: The resulting integrated map contained 661 markers in 13 LGs, with a genomic coverage of 2,774 cM and a mean density of 0.23 markers/cM. The groups were assigned to the nine Citrus haploid chromosomes; however, some of the chromosomes were represented by two LGs due the lack of information for a single integration, as in cases where markers segregated in a 3:1 fashion. A total of 19 QTLs were identified through composite interval mapping (CIM) of the 12 analyzed fruit characteristics: fruit diameter (cm), height (cm), height/diameter ratio, weight (g), rind thickness (cm), segments per fruit, total soluble solids (TSS, %), total titratable acidity (TTA, %), juice content (%), number of seeds, TSS/TTA ratio and number of fruits per box. The genomic sequence (pseudochromosomes) of C. sinensis was compared to the genetic map, and synteny was clearly identified. Further analysis of the map regions with the highest LOD scores enabled the identification of putative genes that could be associated with the fruit quality characteristics. CONCLUSION: An integrated linkage map of Murcott tangor and Pera sweet orange using DArTseq™ molecular markers was established and it was useful to perform QTL mapping of twelve fruit quality traits. The next generation sequences data allowed the comparison between the linkage map and the genomic sequence (pseudochromosomes) of C. sinensis and the identification of genes that may be responsible for phenotypic traits in Citrus. The obtained linkage map was used to assign sequences that had not been previously assigned to a position in the reference genome.

Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely 'Candidatus Liberibacter asiaticus' (Las), 'Candidatus Liberibacter africanus', and 'Candidatus Liberibacter americanus'. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

Laser-induced Fluorescence Spectroscopy (LIFS) for Discrimination of Genetically Close Sweet Orange Accessions ( Citrus sinensis L. Osbeck).

Although there is substantial diversity among cultivated sweet oranges genotypes with respect to morphological, physiological, and agronomic traits, very little variation at DNA level has been observed. It is possible that this low DNA molecular variability is due to a narrow genetic basis commonly observed in this citrus group. The most different morphological characters observed were originated through mutations, which are maintained by vegetative propagation. Despite all molecular tools available for discrimination between these different accessions, in general, low polymorphism has been observed in all groups of sweet oranges and they may not be identified by molecular markers. In this context, this paper describes the results obtained by using laser-induced fluorescent spectroscopy (LIFS) as a tool to discriminate sweet orange accessions ( Citrus sinensis L. Osbeck) including common, low acidity, pigmented, and navel orange groups, with very little variation at DNA level. The findings showed that LIFS combined with statistical methods is capable to discriminate different accessions. The basic idea is that citrus leaves have multiple fluorophores and concentration depends on their genetics and metabolism. Thus, we consider that the optical properties of citrus leaves may be different, depending on variety. The results have shown that the developed method, for the best classification rate, reaches an average sensitivity and specificity of 95% and 97.5%, respectively. An interesting application of this study is the development of an economically viable tool for early identification in seedling certification, in citrus breeding programs, in cultivar protection, or in germplasm core collection.

Incidence of 'Candidatus Liberibacter asiaticus'-Infected Plants Among Citrandarins as Rootstock and Scion Under Field Conditions.

Huanglongbing (HLB), caused by the bacterium 'Candidatus Liberibacter' spp., is currently one of the most serious diseases of citrus plants and has caused substantial economic losses. Thus far, there is no source of genetic resistance to HLB in the genus Citrus or its relatives. However, several studies have reported Poncirus trifoliata and some of its hybrids to be more tolerant to the disease. The main objective of this study was to report differences in the incidence of 'Ca. L. asiaticus' infection in citrandarin plants, hybrids from Sunki mandarin (Citrus sunki (Hayata) hort. ex Tanaka), and trifoliate orange Rubidoux (P. trifoliata (L.) Raf.)), after conducting an extensive survey under field conditions. These hybrid plants were established for approximately 7 years in an area with a high incidence of 'Ca. L. asiaticus'-infected plants. We selected two experimental areas (area A and area B), located approximately 10 m apart. Area A consists of Pera sweet orange (C. sinensis (L.) Osb.) grafted onto 56 different citrandarin rootstocks. Area B consists of citrandarin scions grafted onto Rangpur lime (C. limonia Osb.) rootstock. Bacteria in the leaves and roots were detected using real-time quantitative polymerase chain reaction. The incidence of 'Ca. L. asiaticus'-infected plants was 92% in area A and 14% in area B. Because infected plants occurred in both areas, we examined whether the P. trifoliata hybrid rootstock influenced HLB development and also determined the distribution of 'Ca. L. asiaticus' in Citrus tree tissues. Although this survey does not present evidence regarding the resistance of P. trifoliata and its hybrids in relation to bacteria or psyllids, future investigation, mainly using the most promising hybrids for response to 'Ca. L. asiaticus', will help us to understand the probable mechanism of defense or identifying compounds in P. trifoliata and its hybrids that are very important as strategy to combat HLB. Details of these results are presented and discussed in this article.

Screening of genomic libraries.

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs.

Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica.

BACKGROUND: Gummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R), and four F1 susceptible hybrids (Pool S) to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with P. parasitica. RESULTS: Three pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki and Pool R/Pool S) were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (R gene) such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plants CONCLUSIONS: Twenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. UniGene transcripts were likely to be involved in disease resistance, such as genes potentially involved in secondary metabolite synthesis, intracellular osmotic adjustment, signal transduction pathways of cell death, oxidative burst and defense gene expression. Furthermore, our microarray data suggest another type of resistance in Citrus-Phytophthora interaction conferred by single dominant genes (R gene) since we encountered two previously reported R genes (TIR-NBS-LRR and RPS4) upregulated in the resistant genotypes relative to susceptible. We identified 7 transcripts with homology in other plants but yet unclear functional characterization which are an interesting pool for further analyses and 3 transcripts where no significant similarity was found. This is the first microarray study addressing an evaluation of transcriptional changes in response to P. parasitica in Citrus.

Development of genetic maps of the citrus varieties 'Murcott' tangor and 'Pera' sweet orange by using fluorescent AFLP markers.

The progeny of 87 BC(1) hybrids of 'Murcott' tangor and 'Pera' sweet orange, genotyped with fluorescent amplified fragment length polymorphism (fAFLP) markers, was used for the construction of genetic maps for both citrus varieties. Mapping strategies, considering the progeny as a result of backcrossing and cross-pollination, were exploited in Mapmaker 2.0 (LOD score >or= 3.0 and or= 3.0 and theta

In silico analysis of phytohormone metabolism and communication pathways in citrus transcriptome

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

Differential expression of genes identified from Poncirus trifoliata tissue inoculated with CTV through EST analysis and in silico hybridization

Citrus is the most important fruit crop in Brazil and Citrus tristeza virus (CTV) is considered one of the most important pathogens of citrus. Most citrus species and varieties are susceptible to CTV infection. However, Poncirus trifoliata, a close relative of citrus, is resistant to the virus. In order to better understand the responses of citrus plants to the infection of CTV, we constructed expressed sequence tag (EST) libraries with tissues collected from Poncirus trifoliata plants, inoculated or not with Citrus tristeza virus at 90 days after inoculation, grafted on Rangpur lime rootstocks. We generated 17,867 sequence tags from Poncirus trifoliata inoculated (8,926 reads) and not (8,941 reads) with a severe CTV isolate. A total of 2,782 TCs (Tentative Consensi sequences) were obtained using both cDNA libraries in a single clusterization procedure. By the in silico hybridization approach, 289 TCs were identified as differentially expressed in the two libraries. A total of 121 TCs were found to be overexpressed in plants infected with CTV and were grouped in 12 primary functional categories. The majority of them were associated with metabolism and defense response. Some others were related to lignin, ethylene biosynthesis and PR proteins. In general, the differentially expressed transcripts seem to be somehow involved in secondary plant response to CTV infection.

Frequency and distribution of microsatellites from ESTs of citrus

Nearly 65,000 citrus EST (Expressed Sequence Tags) have been investigated using the CitEST project database. Microsatellites were investigated in the unigene sequences from Citrus spp. and Poncirus trifoliata. From these sequences, approximately 35 percent of the non-redundant ESTs contained SSRs. The frequencies of different SSR motifs were similar between Citrus spp and trifoliate orange. In general, mononucleotide repeats appeared to be the most abundant SSRs in the CitEST database, but we also identify di-, tri-, tetra-, penta- and hexanucleotide repeats. The AG/CT and AAG/CTT were the most common dinucleotide and trinucleotide motifs, with frequencies of 54.4 percent and 25.2 percent, respectively. Primer sequences flanking SSR motifs were successfully designed and synthesized. After in silico polymorphism analysis, a subset of sixty-eight primers was validated in different Citrus spp. and Poncirus trifoliata. PCR-amplification revealed polymorphism in citrus with all tested primer pairs and showed the potential of these markers for linkage mapping. Our study showed that the CitEST database can be exploited for the development of SSR markers that can amplify Citrus spp. and related genus for comparative mapping and other genetic analyses.