Project description:To identify genes associated with citrus peel development and manifestation of peel disorders, we analyzed flavedo, albedo and juice sac tissues from five types of citrus fruit including, mandarin orange, navel orange, valencia orange, grapefruit and lemon. Fruits of five different citrus cultivars. Mature, healthy fruits of five different citrus cultivars (M-bM-^@M-^\ValenciaM-bM-^@M-^] and M-bM-^@M-^\NavelM-bM-^@M-^] orange [Citrus sinensis], mandarin [Citrus reticulata], lemon [Citrus M-CM-^W limon], grapefruit [Citrus M-CM-^W paradisi]) were purchased from a food market located in Davis, CA, USA. For all five types of fruit, three tissues (flavedo, albedo, and juice sacs) were compared separately. Each of the three tissues from each of the five types of fruit were sampled in three biological replicates, for a total of 45 samples. Samples were prepared from a 1 cm-thick equatorial disc and four sections (N, S, E, and W) were cut. Each section of flavedo, albedo, and juice sac tissue was dissected. gene expression variation underlying quality trait, different genotypes
Project description:Huanglongbing (HLB) (=citrus greening) is a destructive disease of citrus which is caused by a fastidious, phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Of the more than 33,000 probe sets on the microarray 21,067 were expressed in the leaves, of which 279 and 515 were differentially expressed (FDR ≤ 0.05) five to nine and 13-17 weeks after inoculation, respectively. Results from semi-quantitative RT-PCR analysis performed on 14 selected genes were highly correlated with those observed with the microarray. Gene expression changes involved a variety of different processes including cell defense, transport, cellular organization, photosynthesis, and carbohydrate metabolism. Notable was the pathogen-induced accumulation of transcripts for a phloem-specific lectin PP2-like protein. Transcriptional changes and their relation to disease symptom development are discussed. This is the first study of transcriptional profiling in citrus in response to liberibacter infection using microarray technology. Huanglongbing (HLB) is a destructive disease of citrus which is suspected to be caused by a phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange (C. sinensis) leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus (Las), the pathogen associated with HLB in Florida, using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Three year-old 'Valencia' orange (C. sinensis) scions on Cleopatra mandarin (C. reticulata Blanco) rootstocks were graft-inoculated with non-infected (control) or Las-infected tissue from greenhouse- (control) or field-grown 'Lisbon' lemon (C. limon) trees. Fully expanded leaves were collected at 5, 9, 13, and 17 weeks after inoculation (wai). Total RNA was extracted from two control plants and from three Las-inoculated plants per time point. Equal amounts of RNA were combined from samples collected 5 wai and 9 wai and from samples collected 13 wai and 17 wai, resulting in two non-infected and three infected biological replicates for both early (5-9 wai) and late (13-17 wai) time points and used for hybridization on Affymetrix citrus microarrays.
Project description:Citrus species are among the most important fruit crops. However, gene regulation and signaling pathways related to etiolation in this crop remain unknown. Using Illumina sequencing technology, modification of global gene expression in two hybrid citrus cultivars—Huangguogan and Shiranuhi, respectively—were investigated. More than 834.16 million clean reads and 125.12 Gb of RNA-seq data were obtained, more than 91.37% reads had a quality score of Q30 (sequencing error rate, 0.1%). 124,952 unigenes were finally generated with a mean length of 1,189 bp. Of these unigenes, 98,904 (79.15%), 105,408 (84.35%), 42,016 (33.62%), 78,872 (63.12%), 72,068 (57.67%), 72,464 (57.99%), 72464 (57.99%) and 46,308 (37.06%) had been annotated in NR, NT, KO, SwissProt, PFAM, GO and KOG databases, respectively. Further, we identified 604 differentially expressed genes (DEGs) in multicoloured and etiolated seedlings of Shiranuhi, including 180 up-regulated genes and 424 down-regulated genes. While in Huangguogan, we found 1,035 DEGs, 271 of which were increasing and the others were decreasing. 7 DEGs were commonly up-regulated, and 59 DEGs down-regulated in multicoloured and etiolated seedlings of these two cultivars, suggesting that some genes play fundamental roles in two hybrid citrus seedlings during etiolation. Functional classification of the DEGs in two cultivars using GO term indicated that biological process, cellular component and molecular function were three major groups. Our study is the first to provide the transcriptome sequence resource for seedlings etiolation of Shiranuhi and Huangguogan, and advance our knowledge of the genes involved in the complex regulatory networks of seedling etiolation.
Project description:To identify miRNAs related nucellar embryo intiation (NEI) in citrus, we performed small RNA sequencing in ovules of two pairs of poly- and mono-embryonic cultivars right prior to and at NEI stages. Nearly 150 miRNAs including ~90 conserved and ~60 novel miRNAs were identified in ovules of each cultivar pair.
Project description:Candidatus Liberibacter asiaticus (Las) is an emergent bacterial pathogen associated with the devastating citrus Huanglongbing (HLB), also known as the greening disease. Vectored by the Asian Citrus Psyllid (Diaphorina citri), Las colonizes the phloem tissue of citrus. So far, efforts of cultivating Las in vitro have not been successful and dual-transcriptome analyses could only detect ~100 Las genes due to the low abundance of bacterial RNA in infected citrus/psyllid tissues. Therefore, the biology of this pathogen is poorly understood. Here, we established a procedure to enrich Las RNA for transcriptome analysis in order to obtain insights into the interactions of Las with its two hosts. We were able to confidently determine the expression profiles of >400 Las genes, including 106 that were differentially expressed between citrus and psyllids. Genes related to transcription/translation and defense were found to be upregulated in citrus; whereas genes upregulated in psyllids are involved in metabolic pathways related to tricarboxylic acid (TCA) cycle. Genes encoding the succinate dehydrogenase and NADH quinone oxidoreductase complexes, as well as the flagellar system are also expressed to higher levels in psyllids. We also analyzed the relative expression levels of Sec-delivered effectors, which are considered key virulence factors of Las. This work advances our understanding of the HLB biology and offers novel insight into the HLB pathogenesis.
Project description:Candidatus Liberibacter asiaticus (Las), a non-culturable phloem-limited bacterium, is the suspected causal agent of Huanglongbing (HLB) in Florida. HLB is one of the most devastating diseases of citrus and no resistant cultivars have been identified to date, though tolerance has been suggested in the genus Poncirus. A recent study conducted in our laboratory demonstrated tolerance of US-897, a hybrid of Poncirus trifoliata (L. Raf.) and the susceptible ‘Cleopatra’ mandarin (Citrus reticulata Blanco), to Ca. L. asiaticus, the presumed causal agent of HLB in Florida (Albrecht & Bowman, HortScience 46 (2011) 16-22). This study compares transcriptional changes in tolerant US-897 and susceptible ‘Cleopatra’ mandarin seedlings in response to infection with Las using the Affymetrix GeneChip citrus array with the main objective of identifying genes associated with tolerance to HLB. Such genes may be suitable as potential targets for biotechnology approaches, providing one strategy to possibly control this destructive disease of citrus. Fifteen months-old seedlings of the genotypes 'Cleopatra' mandarin and US-897 were graft-inoculated with non-infected (control) or Las-infected tissue from greenhouse-grown 'Valencia' (C. sinensis L.) plants. Six non-infected control plants each from 'Cleopatra' (MC) and US-897 (TC) and six infected plants each from Cleopatra (MI) and US-897 (TI) were used. Four to six leaves per plant were excised at 32 weeks after inoculation (wai) and immediately frozen in liquid nitrogen. Total RNA was extracted from all 24 plants. Equal amounts of RNA from two samples were pooled to obtain three biological replicates per genotype and treatment and used for hybridization on Affymetrix citrus microarrays.
Project description:Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp. In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 30,171 sets expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories.
Project description:Nucellar embryony is a form of apomixis found in citrus where somatic nucellar cells differentiate into embryos and are included in the seed resulting from the normal sexual process. The nucellar cells giving rise to adventive embryo start proliferating prior to anthesis and fully differentiate obtaining nourishment from sexually derived endosperm. To identify transcripts differentially expressed during nucellar embryo initiation we have taken RNA samplesfrom different developing stages of ovules from polyembryonic (cv. Vaniglia Sanguigno) and monoembryonic (cv. Temple) cultivars. We used microarray for a detailed analysis of global gene expression during nucellar embryony initiation and development. We have further validated the differentially expressed genes using qRT-PCR. Citrus ovules were harvested from the excised ovaries of different flower developmental stages. These developing stages were decided on the basis of morphological and histological characteristics of flower and female gametophyte, respectively. We selected, Pre anthesis, post anthesis, and initial fruit set stage of flower to excise ovules from Polyembryonic cultivar Vaniglia Sanguigno and Monoembryonic cultivar Temple. Leaf tissue of Vaniglia Sanguigno was used to check the overall transcriptome level differences between leaf and ovule tissues. For each sample, three technical replicates were used.
Project description:The goals of this study are to reveal the transcriptional and regulatory dynamics of the lncRNAs in response to CLas infection and provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation. For the purpose, we obtained RNA-seq-based differential expression profile of the leave midrib of CLas- and mock-inoculated rough lemon and sweet orange at week 0, 7, 17, and 34 following infections with three biological replicates in the greenhouse. Approximately 2,523 million paired-end reads from 48 sample libraries were produced using Illumina HiSeq 2000. we systematically identified lncRNAs from rough lemon and sweet orange at four different time points of a greenhouse experiment and characterized their genomic transcriptional and regulatory dynamics. We predicted their potential regulatory genes and functions and constructed a co-expression network. Our study provides precious information and expands the knowledge of citrus lncRNA against HLB disease.
Project description:Nucellar embryony is a form of apomixis found in citrus where somatic nucellar cells differentiate into embryos and are included in the seed resulting from the normal sexual process. The nucellar cells giving rise to adventive embryo start proliferating prior to anthesis and fully differentiate obtaining nourishment from sexually derived endosperm. To identify transcripts differentially expressed during nucellar embryo initiation we have taken RNA samplesfrom different developing stages of ovules from polyembryonic (cv. Vaniglia Sanguigno) and monoembryonic (cv. Temple) cultivars. We used microarray for a detailed analysis of global gene expression during nucellar embryony initiation and development. We have further validated the differentially expressed genes using qRT-PCR.