[Show abstract][Hide abstract] ABSTRACT: Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH).
Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control.
The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.
[Show abstract][Hide abstract] ABSTRACT: Pummelo (Citrus grandis Osbeck) is one of the ancestors of sweet orange and economically important cultivated species of the family Rutaceae. The availability of the EST SSR markers for Pummelo represents a promising source to increase the number of markers available for the citrus species. In this study, we evaluated 212 Pummelo EST derived SSR markers (CgEMS) for their transferability across the genera, polymorphism, mapping ability and utility for genetic diversity analysis. Among these markers, 136 were amplified bands, 99 were transferable across the genera. Transferability of CgEMS to C. sinensis, C. reticulata, C. lemon, Fortunella sp and Poncirus sp was 76%, 76%, 75%, 74% and 73%, respectively. In contrast 52 (53%) markers were found to be polymorphic and segregating in a mapping population. Segregating markers can be categorized into four groups: full informative (8%), male informative (15%), female informative (19%) and partly informative (59%). The phylogenetic relationship between the citrus and its relatives obtained with CgEMS was in a good agreement with the established citrus taxonomy and phylogeny. CgEMS could potentially serve as perfect markers for determining variation in phenotype, fingerprinting, mapping and genetic diversity study in C. grandis. Their high level of cross genera transferability of the CgEMS markers has allowed phylogenetic inference within the Rutaceae.
Scientia Horticulturae 05/2013; 155:85–91. · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to develop a large set of microsatellite markers based on publicly available BAC-end sequences (BESs), and to evaluate their transferability, discriminating capacity of genotypes and mapping ability in Citrus. A set of 1,281 simple sequence repeat (SSR) markers were developed from the 46,339 Citrus clementina BAC-end sequences (BES), of them 20.67% contained SSR longer than 20 bp, corresponding to roughly one perfect SSR per 2.04 kb. The most abundant motifs were di-nucleotide (16.82%) repeats. Among all repeat motifs (TA/AT)n is the most abundant (8.38%), followed by (AG/CT)n (4.51%). Most of the BES-SSR are located in the non-coding region, but 1.3% of BES-SSRs were found to be associated with transposable element (TE). A total of 400 novel SSR primer pairs were synthesized and their transferability and polymorphism tested on a set of 16 Citrus and Citrus relative's species. Among these 333 (83.25%) were successfully amplified and 260 (65.00%) showed cross-species transferability with Poncirus trifoliata and Fortunella sp. These cross-species transferable markers could be useful for cultivar identification, for genomic study of Citrus, Poncirus and Fortunella sp. Utility of the developed SSR marker was demonstrated by identifying a set of 118 markers each for construction of linkage map of Citrus reticulata and Poncirus trifoliata. Genetic diversity and phylogenetic relationship among 40 Citrus and its related species were conducted with the aid of 25 randomly selected SSR primer pairs and results revealed that citrus genomic SSRs are superior to genic SSR for genetic diversity and germplasm characterization of Citrus spp.
[Show abstract][Hide abstract] ABSTRACT: Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial
plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity
and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants.
In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that
further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration
medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines
were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet
orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled
haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state
of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.
KeywordsCitrus–Anther culture–Doubled haploid–Allelic diversity–Carotenoid biosynthesis
Plant Cell Tissue and Organ Culture 01/2011; 104(3):415-423. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Red-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.
We 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.
This 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.
[Show abstract][Hide abstract] ABSTRACT: Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 22.214.171.124)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.
[Show abstract][Hide abstract] ABSTRACT: The precocious trifoliate orange (Poncirus trifoliata [L.] Raf.), an early flowering mutant of P. trifoliata, has a short juvenile phase of about 14 months, significantly shorter than other citrus. In this report, using the stems of precocious trifoliate orange seedlings as explants, an improved protocol mediated by Agrobacterium tumefaciens was developed to evaluate regeneration and transformation frequency. The transformed plants continued to reveal the precocious trait of non-transgenic mature precocious trifoliate orange, and as such could prove to be an efficient system for the verification of gene function in the fruits/flower via genetic transformation in woody plants.
Scientia Horticulturae 02/2009; 119(3):335-338. · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and used to analyze chloroplast diversity of Citrus and closely related genera. Fourteen cpSSR primer pairs from the chloroplast genomes of tobacco (Nicotiana tabacum L.) and Arabidopsis were found useful for analyzing the Citrus chloroplast genome (cpDNA) and recoded with the prefix SPCC (SSR Primers for Citrus Chloroplast). Eleven of the 14 primer pairs revealed some degree of polymorphism among 34 genotypes of Citrus, Fortunella, Poncirus and some of their hybrids, with polymorphism information content (PIC) values ranging from 0.057 to 0.732, and 18 haplotypes were identified. The cpSSR data were analyzed with NTSYS-pc software, and the genetic relationships suggested by the unweighted pair group method based on arithmetic means (UPGMA) dendrogram were congruent with previous taxonomic investigations: the results showed that all samples fell into seven major clusters, i.e., Citrus medica L., Poncirus, Fortunella, C. ichangensis Blanco, C. reticulata Swingle, C. aurantifolia (Christm.) Swingle and C. grandis (L.) Osbeck. The results of previous studies combined with our cpSSR analyses revealed that: (1) Calamondin (C. madurensis Swingle) is the result of hybridization between kumquat (Fortunella) and mandarin (C. reticulata), where kumquat acted as the female parent; (2) Ichang papeda (C. ichangensis) has a unique taxonomic status; and (3) although Bendiguangju mandarin (C. reticulata) and Satsuma mandarin (C. reticulata) are similar in fruit shape and leaf morphology, they have different maternal parents. Bendiguangju mandarin has the same cytoplasm as sweet orange (C. sinensis), whereas Satsuma mandarin has the cytoplasm of C. reticulata. Seventeen PCR products from SPCC1 and 21 from SPCC11 were cloned and sequenced. The results revealed that mononucleotide repeats as well as insertions and deletions of small segments of DNA were associated with SPCC1 polymorphism, whereas polymorphism generated by SPCC11 was essentially due to the variation in length of the mononucleotide repeats.
Tree Physiology 07/2005; 25(6):661-72. · 3.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a method for rapid isolation of flanking regions from amplified fragment length polymorphism (AFLP) fragments based
on thermal asymmetric interlaced (TAIL)-PCR, in which one sequence-specific primer and one degenerate primer derived from
an conserved motif found in homologies of the known sequence were used. The final result showed this to be a simple and efficient
strategy, especially for short known sequences containing coding regions. Moreover this protocol was especially useful for
species with little available genome information such as Hongkong Kumquat (Fortunella hindsii), since most of their genes have known homologies in other species such asArabidopsis and rice.