De Novo Sequencing and Hybrid Assembly of the Biofuel Crop Jatropha curcas L.: Identification of Quantitative Trait Loci for Geminivirus Resistance.

Jatropha curcas is an important perennial, drought tolerant plant that has been identified as a potential biodiesel crop. We report here the hybrid de novo genome assembly of J. curcas generated using Illumina and PacBio sequencing technologies, and identification of quantitative loci for Jatropha Mosaic Virus (JMV) resistance. In this study, we generated scaffolds of 265.7 Mbp in length, which correspond to 84.8% of the gene space, using Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Additionally, 96.4% of predicted protein-coding genes were captured in RNA sequencing data, which reconfirms the accuracy of the assembled genome. The genome was utilized to identify 12,103 dinucleotide simple sequence repeat (SSR) markers, which were exploited in genetic diversity analysis to identify genetically distinct lines. A total of 207 polymorphic SSR markers were employed to construct a genetic linkage map for JMV resistance, using an interspecific F2 mapping population involving susceptible J. curcas and resistant Jatropha integerrima as parents. Quantitative trait locus (QTL) analysis led to the identification of three minor QTLs for JMV resistance, and the same has been validated in an alternate F2 mapping population. These validated QTLs were utilized in marker-assisted breeding for JMV resistance. Comparative genomics of oil-producing genes across selected oil producing species revealed 27 conserved genes and 2986 orthologous protein clusters in Jatropha. This reference genome assembly gives an insight into the understanding of the complex genetic structure of Jatropha, and serves as source for the development of agronomically improved virus-resistant and oil-producing lines.

Isolation and sequence characterization of DNA-A genome of a new begomovirus strain associated with severe leaf curling symptoms of Jatropha curcas L.

Begomoviruses belong to the family Geminiviridae are associated with several disease symptoms, such as mosaic and leaf curling in Jatropha curcas. The molecular characterization of these viral strains will help in developing management strategies to control the disease. In this study, J. curcas that was infected with begomovirus and showed acute leaf curling symptoms were identified. DNA-A segment from pathogenic viral strain was isolated and sequenced. The sequenced genome was assembled and characterized in detail. The full-length DNA-A sequence was covered by primer walking. The genome sequence showed the general organization of DNA-A from begomovirus by the distribution of ORFs in both viral and anti-viral strands. The genome size ranged from 2844 bp-2852 bp. Three strains with minor nucleotide variations were identified, and a phylogenetic analysis was performed by comparing the DNA-A segments from other reported begomovirus isolates. The maximum sequence similarity was observed with Euphorbia yellow mosaic virus (FN435995). In the phylogenetic tree, no clustering was observed with previously reported begomovirus strains isolated from J. curcas host. The strains isolated in this study belong to new begomoviral strain that elicits symptoms of leaf curling in J. curcas. The results indicate that the probable origin of the strains is from Jatropha mosaic virus infecting J. gassypifolia. The strains isolated in this study are referred as Jatropha curcas leaf curl India virus (JCLCIV) based on the major symptoms exhibited by host J. curcas.

Molecular characterization of a new begomovirus associated with leaf yellow mosaic disease of Jatropha curcas in India.

During a survey in June 2011, severe leaf yellow mosaic disease was observed on about 45 % plants of Jatropha curcas growing in the Katerniaghat wildlife sanctuary in India. An association of a begomovirus with disease was detected in 15 out of 20 samples by PCR using begomovirus genus-specific primers and total DNA isolated from symptomatic leaf samples. For identification of the begomovirus, the complete genome was amplified using a Phi-29 DNA-polymerase-based rolling-circle amplification kit and total DNA from five representative samples and then digested with BamHI. The linearized RCA products were cloned and sequenced. Their GenBank accession numbers are JN698954 (SKRK1) and JN135236 (SKRK2). The sequences of the two begomovirus isolates were 97 % identical to each other and no more than 86 % to those of jatropha mosaic India virus (JMIV, HM230683) and other begomoviruses reported worldwide. In phylogenetic analysis, SKRK1 and SKRK2 clustered together and showed distant relationships to jatropha mosaic India virus, Jatropha curcas mosaic virus, Indian cassava mosaic virus, Sri Lankan cassava mosaic virus and other begomoviruses. Based on 86 % sequence identities and distant phylogenetic relationships to JMIV and other begomoviruses and the begomovirus species demarcation criteria of the ICTV (<89 % sequence identity of complete DNA-A genome), the begomovirus isolates associated with leaf yellow mosaic disease of J. curcas were identified as members of a new begomovirus species and provisionally designated as jatropha leaf yellow mosaic Katerniaghat virus (JLYMKV). Agroinfectious clones of the DNA molecule of the begomovirus isolate were also generated, and the fulfillment of Koch's postulates was demonstrated in J. curcas plants.

Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India.

Severe yellow mosaic disease was observed in three ornamental species of Jatropha: J. integerrima, J. podagrica and J. multifida grown in gardens at Lucknow, India, during a survey in 2013. The causal pathogen was successfully transmitted from diseased to healthy plants of these species by whitefly (Bemisia tabaci). The infection of begomovirus was initially detected in naturally infected plant samples by PCR using begomovirus universal primers. The begomovirus was characterized having a monopartite genome based on sequence analyses of the cloned ∼2.9kb DNA-A genome amplified by rolling circle amplification using Phi-29 DNA polymerase. The genome contained 2844 nucleotides that could be translated into seven potential open reading frames. The nucleotide sequences of DNA-A genome of the begomovirus isolates: JI (KC513823), JP (KF652078) and JM (KF652077) shared 94-95% identities together and 93-95% identities with an uncharacterized begomovirus isolated from J. curcas (the only sequences available in GenBank database as GU451249 and EU798996 under the name jatropha leaf curl virus). These shared highest identity of 61% and highly distant phylogenetic relationships with other begomoviruses reported worldwide. Based on 61% sequence identities (much less than 89%, the species demarcation criteria for a new begomovirus) the isolates under study were identified as members of a new Begomovirus species for which the name was proposed as "Jatropha mosaic Lucknow virus (JMLV)". The recombination analysis also suggested that JMLV was not a recombinant species, hence considered as unidentified Begomovirus species. Koch's postulates were also established by agroinfiltration assay of agroinfectious clone of JMLV. Characterization of JMLV associated with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida is being reported for the first time.

Molecular identification of a new begomovirus associated with leaf crumple disease of Jatropha curcas L. in India.

Association of a new begomovirus with leaf crumple disease of Jatropha curcas was identified based on sequence analysis of cloned ~2.7-kb viral DNA genomes from three representative samples amplified by RCA using phi-29 DNA polymerase. Sequence analysis of these isolates SKJ1 (KM066975), SKJ2 (KM189818) and SKJ3 (KM189819)--showed the presence of a monopartite begomovirus-like genome of 2,735 to 2,738 nucleotides containing seven ORFs: V3, V2 and V1 in virion sense and C3, C2, C1 and C4 in the complementary sense. Pairwise alignment showed 99 % nucleotide sequence similarity to each other and less than 81 % identity to other begomoviruses reported worldwide. Based on begomovirus species demarcation criteria for a new species (sequence identity <89 %), the begomovirus isolates were identified as the members of a new Begomovirus species and provisionally designated as Jatropha leaf crumple India virus (JLCrIV).

Molecular and Biological Characterization of Distinct Strains of Jatropha mosaic virus from the Dominican Republic Reveal a Potential to Infect Crop Plants.

In the Dominican Republic (DO), jatropha plants with yellow mosaic symptoms are commonly observed in and around fields of various crop plants. Complete nucleotide sequences of DNA-A and DNA-B components of four bipartite begomovirus isolates associated with symptomatic jatropha plants collected from three geographical locations in the DO were determined. Sequence comparisons revealed highest identities (91 to 92%) with the DNA-A component of an isolate of Jatropha mosaic virus (JMV) from Jamaica, indicating that the bipartite begomovirus isolates from the DO are strains of JMV. When introduced into jatropha seedlings by particle bombardment, the cloned components of the JMV strains from the DO induced stunting and yellow mosaic, indistinguishable from symptoms observed in the field, thereby fulfilling Koch's postulates for the disease. The JMV strains also induced disease symptoms in Nicotiana benthamiana, tobacco, and several cultivars of common bean from the Andean gene pool, including one locally grown in the DO. Asymmetry in the infectivity and symptomatology of pseudorecombinants provided further support for the strain designation of the JMV isolates from the DO. Thus, JMV in the DO is a complex of genetically distinct strains that have undergone local evolution and have the potential to cause disease in crop plants.

The complete genome sequence of New World jatropha mosaic virus.

Full-length sequences of a bipartite begomovirus were obtained from a plant of Jatropha multifida in Florida showing symptoms of foliar mosaic, distortion and necrosis. Sequences of four clones each of a DNA-A and DNA-B were obtained, which showed very low sequence diversity among themselves. The clones were infectious when biolistically inoculated to J. multifida, Phaseolus vulgaris and Nicotiana tabacum, but not to J. curcas. The DNA-A sequences had less than 89 % pairwise identity scores with the DNA-A of other begomoviruses. The DNA-A appeared to be a recombinant in that 18 % of the DNA-A (470 nt) had a pairwise identity score of 91.98 % with RhRGMV, indicating that this portion most likely originated from a virus closely related to RhRGMV. The remaining 82 % of the DNA-A had lower identity scores with TbMoLCV (87.84 %) and RhRGMV (87.46 %), which suggests that this part of the component originated from an undescribed virus. There was no evidence for recombination in the DNA-B. Equivalent sequences of the DNA-A had the highest identity score (94.18 %) with a 533-nt sequence obtained from J. multifida from Puerto Rico in 2001 (GenBank accession no. AF058025). Pairwise comparison, recombination and phylogenetic analysis, and biology suggest that these clones are those of jatropha mosaic virus first reported from Puerto Rico. This is the first report of the complete genome sequence of jatropha mosaic virus.

Virus versus host plant microRNAs: who determines the outcome of the interaction?

Considering the importance of microRNAs (miRNAs) in the regulation of essential processes in plant pathogen interactions, it is not surprising that, while plant miRNA sequences counteract viral attack via antiviral RNA silencing, viruses in turn have developed antihost defense mechanisms blocking these RNA silencing pathways and establish a counter-defense. In the current study, computational and stem-loop Reverse Transcription - Polymerase Chain Reaction (RT-PCR) approaches were employed to a) predict and validate virus encoded mature miRNAs (miRs) in 39 DNA-A sequences of the bipartite genomes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) isolates, b) determine whether virus encoded miRs/miRs* generated from the 5'/3' harpin arms have the capacity to bind to genomic sequences of the host plants Jatropha or cassava and c) investigate whether plant encoded miR/miR* sequences have the potential to bind to the viral genomes. Different viral pre-miRNA hairpin sequences and viral miR/miR* length variants occurring as isomiRs were predicted in both viruses. These miRNAs were located in three Open Reading Frames (ORFs) and in the Intergenic Region (IR). Moreover, various target genes for miRNAs from both viruses were predicted and annotated in the host plant genomes indicating that they are involved in biotic response, metabolic pathways and transcription factors. Plant miRs/miRs* from conserved and highly expressed families were identified, which were shown to have potential targets in the genome of both begomoviruses, representing potential plant miRNAs mediating antiviral defense. This is the first assessment of predicted viral miRs/miRs* of ACMV and EACMV-UG and host plant miRNAs, providing a reference point for miRNA identification in pathogens and their hosts. These findings will improve the understanding of host- pathogen interaction pathways and the function of viral miRNAs in Euphorbiaceous crop plants.

First report of a complete genome sequence for a begomovirus infecting Jatropha gossypifolia in the Americas.

Jatropha gossypifolia is a weed that is commonly found with yellow mosaic symptoms growing along the roadside and in close proximity to cultivated crops in many farming communities in Jamaica. For the first time, the complete genome sequence of a new begomovirus, designated jatropha mosaic virus-[Jamaica:Spanish Town:2004] (JMV-[JM:ST:04]), was determined from field-infected J. gossypifolia in the western hemisphere. DNA-A nucleotide sequence comparisons showed closest identity (84 %) to two tobacco-infecting viruses from Cuba, tobacco mottle leaf curl virus-[Cuba:Sancti Spiritus:03] (TbMoLCV-[CU:SS:03]) and tobacco leaf curl Cuba virus-[Cuba:Taguasco:2005] (TbLCuCUV-[CU:Tag:05]), and two weed-infecting viruses from Cuba and Jamaica, Rhynchosia rugose golden mosaic virus-[Cuba:Camaguey:171:2009] (RhRGMV- [CU:Cam:171:09]) and Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). Phylogenetic analysis revealed that JMV-[JM:ST:04] is most closely related to tobacco and tomato viruses from Cuba and WGMSTV-[JM:Alb:05], a common malvaceous-weed-infecting virus from eastern Jamaica, and that it is distinct from begomoviruses infecting Jatropha species in India and Nigeria.

DNA-A of a highly pathogenic Indian cassava mosaic virus isolated from Jatropha curcas causes symptoms in Nicotiana benthamiana.

Jatropha curcas mosaic disease (JcMD) is a newly emerging disease that has been reported in Africa and India. Here, we report the complete nucleotide sequence of a new Indian cassava mosaic virus isolate (ICMV-SG) from Singapore. Infection of ICMV-SG showed more severe JcMD in Jatropha curcas and Nicotiana benthamiana than the other ICMV isolates reported previously, though ICMV-SG shares high sequence identity with the other ICMV isolates. Agroinfectious DNA-A alone sufficiently induced systemic symptoms in N. benthamiana, but not in J. curcas. Results from agroinfection assays showed that systemic infection of ICMV-SG in J. curcas required both DNA-A and DNA-B components.

Molecular identification of a new begomovirus associated with mosaic disease of Jatropha curcas L. in Nigeria.

We report the complete nucleotide sequence of DNA-A of a begomovirus naturally infecting Jatropha curcas L. in Nigeria. Symptoms observed on infected plants were severe mosaic, mottling and blistering of leaves. The virus, which we provisionally name "jatropha mosaic Nigeria virus" (JMNV), has a monopartite genome of 2,779 to 2,789 nucleotides. Pairwise comparisons of DNA-A sequences showed that JMNV had maximum nucleotide sequence identity (72%) with a strain of tomato yellow leaf curl virus. Since there are widespread infections of jatropha in Nigeria showing similar symptoms as those investigated in the present study, JMNV may represent a significant threat to a promising bioenergy crop.

Molecular identification of a new begomovirus associated with yellow mosaic disease of Jatropha gossypifolia in India.

Yellow mosaic disease was observed on Jatropha gossypifolia plants growing in Kathaupahadi, Madhya Pradesh, India, and whiteflies (Bemisia tabaci) were found in the vicinity. Association of a new begomovirus with yellow mosaic disease of J. gossypifolia has been detected by PCR using begomovirus DNA-A-specific primers. The complete DNA-A genome (~2.7 kb) of this virus isolate was amplified by rolling-circle amplification (RCA) followed by digestion with Bam HI. The ~2.7-kb amplicons was cloned and sequenced, and the data obtained were submitted to GenBank under accession numbers FJ177030. The genome of the virus isolate consisted of six open reading frames (ORFs): V2 (pre-coat protein) and V1 (coat protein) in the virion sense and C3 (REn protein), C2 (TrAP protein), C1 (replication-associated protein) and C4 (C4 protein) in the complementary sense. BLASTn analysis of the nucleotide sequence (2757 nt) of the viral genome (FJ177030) showed 84-85% identity and a distinct phylogenetic relationship with DNA-A of tomato leaf curl virus-Bangalore II (U38239) and tomato leaf curl Karnataka virus (AY754812). Based on its 85% sequence identity to all other begomoviruses known to date and ICTV species demarcating criteria (< 88% identity), the name Jatropha yellow mosaic India virus (JYMIV) is proposed. JYMIV is considered to be monopartite, as neither DNA-B nor DNA-ß components associated with begomoviruses were detected.

Biotechnological approaches to determine the impact of viruses in the energy crop plant Jatropha curcas.

BACKGROUND: Geminiviruses infect a wide range of plant species including Jatropha and cassava both belonging to family Euphorbiaceae. Cassava is traditionally an important food crop in Sub - Saharan countries, while Jatropha is considered as valuable biofuel plant with great perspectives in the future. RESULTS: A total of 127 Jatropha samples from Ethiopia and Kenya and 124 cassava samples from Kenya were tested by Enzyme-Linked Immunosorbent Assay (ELISA) for RNA viruses and polymerase chain reaction for geminiviruses. Jatropha samples from 4 different districts in Kenya and Ethiopia (analyzed by ELISA) were negative for all three RNA viruses tested: Cassava brown streak virus (CBSV), Cassava common mosaic virus, Cucumber mosaic virus, Three cassava samples from Busia district (Kenya) contained CBSV. Efforts to develop diagnostic approaches allowing reliable pathogen detection in Jatropha, involved the amplification and sequencing of the entire DNA A molecules of 40 Kenyan isolates belonging to African cassava mosaic virus (ACMV) and East African cassava mosaic virus - Uganda. This information enabled the design of novel primers to address different questions: a) primers amplifying longer sequences led to a phylogenetic tree of isolates, allowing some predictions on the evolutionary aspects of Begomoviruses in Jatrophia; b) primers amplifying shorter sequences represent a reliable diagnostic tool. This is the first report of the two Begomoviruses in J. curcas. Two cassava samples were co - infected with cassava mosaic geminivirus and CBSV. A Defective DNA A of ACMV was found for the first time in Jatropha. CONCLUSION: Cassava geminiviruses occurring in Jatropha might be spread wider than anticipated. If not taken care of, this virus infection might negatively impact large scale plantations for biofuel production. Being hosts for similar pathogens, the planting vicinity of the two crop plants needs to be handled carefully.

Complete nucleotide sequence of Croton yellow vein mosaic virus and DNA-ß associated with yellow vein mosaic disease of Jatropha gossypifolia in India.

A severe yellow vein mosaic disease was noticed on several Jatropha gossypifolia plants growing nearby agriculture fields at Lucknow, India. Diseased plants exhibited yellow vein mosaic, leaf deformation, vein swelling and stunting. A population of whiteflies (Bemisia tabaci) was also noticed in the vicinities; therefore, begomovirus infection was suspected. To confirm begomovirus association, total DNA was isolated from symptomatic leaf samples and subjected to PCR using DNA-A, DNA-B and DNA-ß-specific primers. DNA-A and DNA-ß was successfully amplified but several attempts failed to amplify DNA-B indicating monopartite nature of the begomovirus. The sequence analysis of amplicons revealed the presence of 2757 nucleotides of DNA-A genome (EU727086) and 1315 nt of DNA-ß molecule (EU604296). The sequence analysis of DNA-A (EU727086) revealed the highest 96% identities and closest relationship with Croton yellow vein mosaic virus (CYVMV, AJ507777) infecting Croton bonplandianum in India. The DNA-ß (EU604296) showed the highest 96% sequence identity and closest phylogenetic relationship with CYVMV-associated DNA-ß (AM410551) isolated from Croton sp. in Pakistan. Based on the highest sequence identities and closest phylogenetic relationships of the DNA-A genome and DNA-ß molecule with respective sequences of various isolates of Croton yellow vein mosaic virus, the begomovirus associated with yellow vein mosaic disease of J. gossypifolia was identified as an isolate of Croton yellow vein mosaic virus.

Metabolic and histopathological alterations of Jatropha mosaic begomovirus-infected Jatropha curcas L. by HR-MAS NMR spectroscopy and magnetic resonance imaging.

Alterations in the anatomical structures, sap translocation and metabolic profiles in Jatropha curcas L. (Euphorbiaceae), infected with Jatropha mosaic virus (JMV) have been investigated using MRI and HR-MAS NMR spectroscopy. The contrast of MRI images distinguishes abnormalities in anatomical structures of infected and healthy stem. The HR-MAS NMR spectroscopic analysis indicated that viral infection significantly affected the plant metabolism. Higher accumulation of TCA cycle intermediates, such as citrate and malate, in JMV-infected plants suggested a higher rate of respiration. The respiration rate was more than twofold as compared to healthy ones. The viral stress also significantly increases the concentrations of alanine, arginine, glutamine, valine, GABA and choline as compared to healthy ones. Microscopic examination revealed severe hyperplasia caused by JMV with a considerable reduction in the size of stem cells. Lower concentration of glucose and sucrose in viral-infected stem tissues indicates decreased translocation of photosynthates from leaves to stem due to hyperplasia caused by JMV. The MR images distinguished stele, cortical and pith regions of JMV-infected and healthy stems. Contrast of T(1)- and T(2)-weighted images showed significant differences in the spatial distribution of water, lipids and macromolecules in virus-infected and healthy stem tissues. The results demonstrated the value of MRI and HR-MAS NMR spectroscopy in studying viral infection and metabolic shift in plants. The present methodology may help in better understanding the metabolic alterations during biotic stress in other plant species of agricultural and commercial importance.

A new strain of Indian cassava mosaic virus causes a mosaic disease in the biodiesel crop Jatropha curcas.

Jatropha curcas mosaic disease is a newly emerging disease that challenges the productivity of a prospective biofuel crop, J. curcas. The aetiology of this disease has not been resolved. Here, we report the complete nucleotide sequences of a Jatropha virus isolated from Dharwad, Southern India. Phylogenetic analysis of the virus genome suggests it is a new strain of Indian cassava mosaic virus. Agroinfiltration of the two cloned viral DNA components produced systemic infection and typical mosaic symptoms in J. curcas, thereby fulfilling Koch's postulates. The availability of infectious clones will provide a valuable tool to screen J. curcas cultivars for disease resistance and facilitate the generation of virus-resistant J. curcas plants by transgenic technology.