Wen-Wu Guo

The draft genome of sweet orange (Citrus sinensis)

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

An efficient protocol for genomic DNA extraction fromCitrus species

We describe a simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet easily dissolved in TE buffer. Using this method, DNA was extracted from samples of more than 1000Citrus spp., including young leaves, old leaves, frosted old leaves, withered old leaves, and callus lines. The average yield of DNA ranged from 50–500 μg/g of sample. DNA was suitable for PCR and RFLP analyses and long-term storage. Recently, the procedure was used to isolate DNA from withered old leaves of more than 20 tropical and subtropical fruit crops.

Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type

BackgroundRed-flesh fruit is absent from common sweet orange varieties, but is more preferred by consumers due to its visual attraction and nutritional properties. Our previous researches on a spontaneous red-flesh mutant revealed that the trait is caused by lycopene accumulation and is regulated by both transcriptional and post-transcriptional mechanisms. However, the knowledge on post-transcriptional regulation of lycopene accumulation in fruits is rather limited so far.ResultsWe used Illumina sequencing method to identify and quantitatively profile small RNAs on the red-flesh sweet orange mutant and its wild type. We identified 85 known miRNAs belonging to 48 families from sweet orange. Comparative profiling revealed that 51 known miRNAs exhibited significant expression differences between mutant (MT) and wild type (WT). We also identified 12 novel miRNAs by the presence of mature miRNAs and corresponding miRNA*s in the sRNA libraries. Comparative analysis showed that 9 novel miRNAs are differentially expressed between WT and MT. Target predictions of the 60 differential miRNAs resulted 418 target genes in sweet orange. GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in transcription regulation, protein modification and photosynthesis. The expression profiles of target genes involved in carotenogenesis and photosynthesis were further confirmed to be complementary to the profiles of corresponding miRNAs in WT and MT.ConclusionThis study comparatively characterized the miRNAomes between the red-flesh mutant and the wild type, the results lay a foundation for unraveling the miRNA-mediated molecular processes that regulate lycopene accumulation in the sweet orange red-flesh mutant.

Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange

Somatic embryogenesis (SE) is a remarkable process of plant somatic cells developing into an embryo capable of forming a complete plant. MiRNAs play important roles in plant development by regulating expression of their target genes, but its function in SE has rarely been studied. Herein, ten conserved miRNAs with critical functions in plant development are detected by stem-loop qRT-PCR in the SE system of Valencia sweet orange. Sixteen unigenes from citrus are predicted to be targeted by six of the miRNAs. Cleavage sites on 15 of these target mRNAs are mapped by 5′RACE, of which ten are reported in this study. Transcript abundances of the 16 target unigenes are detected by qRT-PCR during SE process. Stage and tissue-specific expressions of miRNAs and their targets suggest their possible modulation on SE of citrus callus: miR156, 168 and 171 exert regulatory function during somatic embryo induction process; miR159, 164, 390 and 397 are related to globular-shaped embryo formation; miR166, 167 and 398 are required for cotyledon-shaped embryo morphogenesis; in addition, target genes of miR164, 166 and 397 are associated with SE disability of nonembryogenic callus. Exploration of miRNA-mediated modulation on SE is expected to provide new insights into plant cell totipotency, as well as how to maintain and enhance the embryogenic capacity of somatic cells.

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.

Protoplast transformation and regeneration of transgenic valencia sweet orange plants containing a juice quality-related pectin methylesterase gene

Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.

Erratum to: Identification of miRNAs and Their Target Genes Using Deep Sequencing and Degradome Analysis in Trifoliate Orange [Poncirus trifoliata L. Raf]

To identify novel as well as conserved miRNAs in citrus, deep sequencing of small RNA library combined with microarray was performed in precocious trifoliate orange (an early flowering mutant of trifoliate orange, Poncirus trifoliata L. Raf.), resulting in the obtainment of a total of 114 conserved miRNAs belonging to 38 families and 155 novel miRNAs. The miRNA star sequences of 39 conserved miRNAs and 27 novel miRNAs were also discovered among newly identified miRNAs, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 172 and 149 genes were identified as targets of conserved miRNAs and novel miRNAs, respectively. GO and KEGG annotation revealed that high ranked miRNA-target genes were those implicated in biological and metabolic processes. To characterize those miRNAs expressed at the juvenile and adult development stages of citrus, further analysis on the expression profiles of these miRNAs through hybridizing the commercial microarray and real-time PCR was performed. The results revealed that some miRNAs were down-regulated at adult stage compared with juvenile stage. Detailed comparison of the expression patterns of some miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated.

Comparative Transcript Profiling of a Male Sterile Cybrid Pummelo and Its Fertile Type Revealed Altered Gene Expression Related to Flower Development

Male sterile and seedless characters are highly desired for citrus cultivar improvement. In our breeding program, a male sterile cybrid pummelo, which could be considered as a variant of male fertile pummelo, was produced by protoplast fusion. Herein, ecotopic stamen primordia initiation and development were detected in this male sterile cybrid pummelo. Histological studies revealed that the cybrid showed reduced petal development in size and width, and retarded stamen primordia development. Additionally, disorganized cell proliferation was also detected in stamen-like structures (fused to petals and/or carpel). To gain new insight into the underlying mechanism, we compared, by RNA-Seq analysis, the nuclear gene expression profiles of floral buds of the cybrid with that of fertile pummelo. Gene expression profiles which identified a large number of differentially expressed genes (DEGs) between the two lines were captured at both petal primordia and stamen primordia distinguishable stages. For example, nuclear genes involved in nucleic acid binding and response to hormone synthesis and metabolism, genes required for floral bud identification and expressed in particular floral whorls. Furthermore, in accordance with flower morphology of the cybrid, expression of PISTILLATA (PI) was reduced in stamen-like structures, even though it was restricted to correct floral whorls. Down-regulated expression of APETALA3 (AP3) coincided with that of PI. These finding indicated that, due to their whorl specific effects in flower development, citrus class-B MADS-box genes likely constituted ‘perfect targets’ for CMS retrograde signaling, and that dysfunctional mitochondria seemed to cause male sterile phenotype in the cybrid pummelo.

Wide somatic hybrids of Citrus with its related genera and their potential in genetic improvement

Citrus wild relatives are an untapped germplasm reservoir, which possesses many elite resistance traits. Genetic introgression into Citrus by conventional methods has been greatly hindered by polyembryony, pollen / ovule sterility, sexual / graft incompatibility, long juvenility etc. Somatic hybridization via protoplast fusion may make it possible to transfer genes from wild relatives to Citrus. To date, more than sixty sexually compatible or incompatible intergeneric somatic hybrids between Citrus and its various related wild genera have been produced by PEG - orelectrically - induced fusion. These wide somatic hybrids were identified by morphology, cytology, isozyme, RAPD and RFLP analyses. Genetic variation or recombination was also revealed in some of them. Several sexually compatible combinations have flowered and set fruits. The agronomic performance of these wide somatic hybrids is diverse, and the current results are not conclusive. Somatic hybrids are being tested as rootstocks. The prospects of wide somatic hybridization of Citrus with its wild relatives are discussed.

Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing

BackgroundAfter several years in the juvenile phase, trees undergo flowering transition to become mature (florally competent) trees. This transition depends on the balanced expression of a complex network of genes that is regulated by both endogenous and environmental factors. However, relatively little is known about the molecular processes regulating flowering transition in woody plants compared with herbaceous plants.ResultsComparative transcript profiling of spring shoots after self-pruning was performed on a spontaneously early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) with a short juvenile phase and the wild-type (WT) tree by using massively parallel signature sequencing (MPSS). A total of 16,564,500 and 16,235,952 high quality reads were obtained for the WT and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed genes in the MT (31,468) was larger than in the WT (29,864), suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts revealed that 2735 genes had more than twofold expression difference in the MT compared with the WT. In addition, we identified 110 citrus flowering-time genes homologous with known elements of flowering-time pathways through sequencing and bioinformatics analysis. These genes are highly conserved in citrus and other species, suggesting that the functions of the related proteins in controlling reproductive development may be conserved as well.ConclusionOur results provide a foundation for comparative gene expression studies between WT and precocious trifoliate orange. Additionally, a number of candidate genes required for the early flowering process of precocious trifoliate orange were identified. These results provide new insight into the molecular processes regulating flowering time in citrus.