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Tatter leaf disease caused by citrus tatter leaf virus (CTLV) is a serious threat to the citrus industry. In this study, a reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) assay for detection of CTLV was established by optimizing the reaction conditions. The dynamic range of the detection of CTLV RNA transcripts by RT-ddPCR was from 5 to 5 × 105 copies/μL, and the sensitivity was 10 times higher than that of RT real-time PCR. Both techniques showed a high degree of linearity and quantitative correlation. The number of positive samples that was identified in field citrus samples using RT-ddPCR (62.3%) was higher than that of RT-qPCR (49.2%). The results demonstrated that the RT-ddPCR assay is a promising tool for improving CTLV detection.

Virus induced gene silencing (VIGS) is a powerful tool for identifying gene functions in plants. Tobacco rattle virus (TRV)-mediated VIGS has been reported for silencing of endogenous genes in many plants. In citrus, analysis of gene function is mainly relied on the table genetic transformation, even though this technique is low-efficiency and time-consuming. In this study, a TRV-mediated VIGS was successfully applied for verification of gene function in citrus. A reporter gene, Magnesium chelatase subunit I (ChlI) from C. grandis was constructed into TRV2 vector to induce gene silencing with visible photobleaching symptom. TRV VIGS induced photobleaching symptom was observed on the leaves of Nicotiana benthamiana and Citrus after infiltration. The susceptibility to VIGS vector was found to be genotype dependent. Mexican lemon is the most susceptible one. The chlorosis area per leaf was 65.5% of the whole leaf area. GXSTY pummelo exhibited less susceptible to VIGS with only tiny patches of chlorosis appeared on the leaves of treated plants. The parameters were optimized to improve the efficiency. The tiny young leaves and half expanded leaves were more susceptible to VIGS infection. Young seedlings were highly susceptible to vacuum infiltration. The expression of fluorescence from GFP reporter gene was observed in the young seedlings of GXSTY after infiltration with TRV. Close to 100% of infiltration efficiency was achieved by vacuum infiltration. The results indicated that TRV mediated VIGS with vacuum infiltration could be an ideal tool for gene function studies in citrus. A TRV mediated VIGS has been optimized to use in the citrus for verification of gene function. The sensitivity to VIGS vector was genotype dependent. Young seedling and young leaf were the suitable materials for VIGS infection. Vacuum infiltration was a high efficiency infiltration method.

Background Limonoids are major bioactive compounds that are produced by the triterpenoid metabolic pathway. The detailed biochemical process of limonoid biosynthesis and the mechanism of its molecular regulation remain elusive. The identification of transcription factors that regulate limonoid biosynthetic pathways is very important for understanding the underlying regulatory mechanisms. This information could also provide tools for manipulating biosynthesis genes to modulate limonoid production. Results In this study, the CiMYB42 transcription factor was isolated to identify its role in limonoid biosynthesis. Multiple alignment analysis and phylogenetic analysis demonstrated that CiMYB42 is a typical R2R3MYB transcription factor that shares high similarity of its amino acid sequence with AtMYB42. Limonoids contents were higher in Citrus sinensis and Citrus grandis than in other species. Limonoid accumulation during leaf development also showed diverse trends in different genotypes. The expression of CiMYB42 was significantly related to the limonoid content and the expression of CiOSC in some citrus accessions. The overexpression of CiMYB42 in sweet orange resulted in significant accumulation of limonin, whereas the downregulation of CiMYB42 by RNAi resulted in a dwarf phenotype and less nomilin accumulation. Furthermore, the results of a yeast one-hybrid assay and EMSA indicated that CiMYB42 binds exclusively to the TTGTTG sequence (type II MYB core) in the promoter of CiOSC. Together, these results suggest that CiMYB42 positively regulates limonoid biosynthesis by regulating the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core) of its promoter. Conclusions CiMYB42 is an important transcription activator involved in limonoid biosynthesis that regulates the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core).

Background Limonoids are major bioactive compounds that are produced by the triterpenoid metabolic pathway. The detailed biochemical process of limonoid biosynthesis and the mechanism of its molecular regulation remain elusive. The identification of transcription factors that regulate limonoid biosynthetic pathways is very important for understanding the underlying regulatory mechanisms. This information could also provide tools for manipulating biosynthesis genes to modulate limonoid production. Results In this study, the CiMYB42 transcription factor was isolated to identify its role in limonoid biosynthesis. Multiple alignment analysis and phylogenetic analysis demonstrated that CiMYB42 is a typical R2R3MYB transcription factor that shares high similarity of its amino acid sequence with AtMYB42. Limonoids contents were higher in Citrus sinensis and Citrus grandis than in other species. Limonoid accumulation during leaf development also showed diverse trends in different genotypes. The expression of CiMYB42 was significantly related to the limonoid content and the expression of CiOSC in some citrus accessions. The overexpression of CiMYB42 in sweet orange resulted in significant accumulation of limonin, whereas the downregulation of CiMYB42 by RNAi resulted in a dwarf phenotype and less nomilin accumulation. Furthermore, the results of a yeast one-hybrid assay and EMSA indicated that CiMYB42 binds exclusively to the TTGTTG sequence (type II MYB core) in the promoter of CiOSC. Together, these results suggest that CiMYB42 positively regulates limonoid biosynthesis by regulating the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core) of its promoter. Conclusions CiMYB42 is an important transcription activator involved in limonoid biosynthesis that regulates the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core).

In 2009, a new viral disease of citrus caused by citrus yellow vein clearing virus (CYVCV) was first discovered in China. CYVCV is considered to be the most serious pathogen affecting lemon production. In this study, a sensitive and reliable reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay was developed to detect and quantify CYVCV without references. The specificity of the assay was demonstrated by its failure to amplify other relevant citrus viruses. The quantitative linearity, sensitivity and accuracy of RT-ddPCR for detecting CYVCV were compared to those of real-time RT-PCR. The results showed that both methods had a high degree of linearity (R2 = 0.9776) and quantitative correlation. Furthermore, RT-ddPCR was found to be 100 times more sensitive than real-time RT-PCR, and it can therefore be used to detect CYVCV in individual arthropods. In summary, the results demonstrated that the RT-ddPCR assay is a promising approach for quantitative detection of CYVCV with high precision and accuracy.

Background Citrus flavonoids are considered as the important secondary metabolites because of their biological and pharmacological activities. Chalcone synthase (CHS) is a key enzyme that catalyses the first committed step in the flavonoid biosynthetic pathway. CHS genes have been isolated and characterized in many plants. Previous studies indicated that CHS is a gene superfamily. In citrus, the number of CHS members and their contribution to the production of flavonoids remains a mystery. In our previous study, the copies of CitCHS2 gene were found in different citrus species and the sequences are highly conserved, but the flavonoid content varied significantly among those species. Results From seventy-seven CHS and CHS-like gene sequences, ten CHS members were selected as candidates according to the features of their sequences. Among these candidates, expression was detected from only three genes. A predicted CHS sequence was identified as a novel CHS gene. The structure analysis showed that the gene structure of this novel CHS is very similar to other CHS genes. All three CHS genes were highly conserved and had a basic structure that included one intron and two exons, although they had different expression patterns in different tissues and developmental stages. These genes also presented different sensitivities to methyl jasmonate (MeJA) treatment. In transgenic plants, the expression of CHS genes was significantly correlated with the production of flavonoids. The three CHS genes contributed differently to the production of flavonoids. Conclusion Our study indicated that CitCHS is a gene superfamily including at least three functional members. The expression levels of the CHS genes are highly correlated to the biosynthesis of flavonoids. The CHS enzyme is dynamically produced from several CHS genes, and the production of total flavonoids is regulated by the overall expression of CHS family genes. Electronic supplementary material The online version of this article (10.1186/s12870-018-1418-y) contains supplementary material, which is available to authorized users.

Limonoids produced by citrus are a group of highly bioactive secondary metabolites which provide health benefits for humans. Currently there is a lack of information derived from research on the genetic mechanisms controlling the biosynthesis of limonoids, which has limited the improvement of citrus for high production of limonoids. In this study, the transcriptome sequences of leaves, phloems and seeds of pummelo (Citrus grandis (L.) Osbeck) at different development stages with variances in limonoids contents were used for digital gene expression profiling (DGE) analysis in order to identify the genes corresponding to the biosynthesis of limonoids. Pair-wise comparison of transcriptional profiles between different tissues identified 924 differentially expressed genes commonly shared between them. Expression pattern analysis suggested that 382 genes from three conjunctive groups of K-means clustering could be possibly related to the biosynthesis of limonoids. Correlation analysis with the samples from different genotypes, and different developing tissues of the citrus revealed that the expression of 15 candidate genes were highly correlated with the contents of limonoids. Among them, the cytochrome P450s (CYP450s) and transcriptional factor MYB demonstrated significantly high correlation coefficients, which indicated the importance of those genes on the biosynthesis of limonoids. CiOSC gene encoding the critical enzyme oxidosqualene cyclase (OSC) for biosynthesis of the precursor of triterpene scaffolds was found positively corresponding to the accumulation of limonoids during the development of seeds. Suppressing the expression of CiOSC with VIGS (Virus-induced gene silencing) demonstrated that the level of gene silencing was significantly correlated to the reduction of limonoids contents. The results indicated that the CiOSC gene plays a pivotal role in biosynthesis of limonoids.

Limonoids are the important secondary metabolites in the citrus. In this study, the accumulation of limonoids at different fruit developmental stages and distribution among different genotypes, tissues and developmental stages were investigated in 12 pummelo varieties. The large variations on limonoids concentration were found among different varieties, which ranged from 233.78 mg/kg FW to 4090.41 mg/kg FW in the seeds at full color stage of the fruit. Classification of pummelos based on the limonoids content divided 12 varieties into three groups. It was matched well with the geographic origination of the pummelo varieties, suggesting that the accumulation of limonoids was mainly determined by the genotype of the pummelo. Accumulation of the limonoids in different tissues was highly variable, and in a tissue specific fashion. The trend of the change on the levels of nomilin and limonin in the seeds and segment membrane were corresponded to the physiological development of the fruit. The rapid accumulation of nomilin and limonoids was observed from the physiological ripening of the seeds. It suggested that physiological maturation of the seeds is a key point that the seeds accelerate the accumulation of nomilin and limonin. In most of pummelo varieties, 10% color break of the fruit was a phenotypic landmark associated with the maximum level of nomilin accumulated in the seeds.

The polymorphic markers of nuclear SSR (simple sequence repeat), SNP (single nucleotide polymorphism)/InDel (insertion–deletion length polymorphism), and cpSSR (chloroplast SSR) were used to evaluate the genetic diversity of 52 trifoliate oranges and their relatives. The two genomic DNA-based markers produced high values of PIC (polymorphic information content) at 0.45 for SNP/InDel and 0.32 for nuclear SSR, indicating that there is a large diversity of genetic variation within the trifoliate oranges. Three UPGMA (unweighted pair-group method with arithmetic means) dendrograms were generated based on the results of three marker systems. At 0.76 of coefficient, all of Poncirus accessions were clustered into one group, except the 74-1 Early Poncirus, one of the three precocious trifoliate oranges, which was placed separately into another group by nuclear SSR. At a higher coefficient, the trifoliate oranges were discriminated into 4, 6 and 6 groups by nuclear SSR (0.86), SNP/InDel (0.95), and cpSSR (0.95), respectively. The results revealed that three precocious trifoliate oranges were originated from different sources. Hubei Early Poncirus and Beibei Early Poncirus were the asexual mutants of different trifoliate oranges, and 74-1 was probably a sexual hybrid. The present study also indicated that ‘Zhiwenzhou,’ a diploid chimera of trifoliate orange and Satsuma is more closely related to Satsuma rather than to trifoliate orange, which is in concordance with the results of morphological observation.