Identification of the 'Haryejosaeng' mandarin cultivar by multiplex PCR-based SNP genotyping.

Most satsuma mandarin (Citrus unshiu Marc.) cultivars are difficult to identify in the seedling stage based only on morphological traits. Therefore, simple polymerase chain reaction (PCR)-based single-nucleotide polymorphism (SNP) markers were developed to specifically and rapidly distinguish the 'Haryejosaeng' cultivar, which is generally supplied to breeders of other satsuma mandarin cultivars. SNP markers were verified using high-resolution melt (HRM)-specific primers. PCR was performed to distinguish 'Haryejosaeng' from eight other satsuma mandarin cultivars using six SNP markers (P1-P6) specific for 'Haryejosaeng', with one negative control SNP primer pair. The best results were obtained using three SNP markers (P1, P2, and P5). In the multiplex PCR, markers P1, P2, and P5 yielded 165-, 150-, and 526-base pair amplicons, respectively, in 'Haryejosaeng', distinguishing it from other satsuma mandarin cultivars. The selected SNP markers were validated by HRM with HRM-specific primers. The multiplex PCR with P1/P5 and P2/P5 also identified 'Haryejosaeng' obtained from a farm growing 17 different cultivars of satsuma mandarin. Specific SNP molecular markers were determined for accurately identifying the 'Haryejosaeng' cultivar by multiplex PCR to save the time and costs associated with its supply to breeders of satsuma mandarin.

Overexpression of Acyl-ACP Thioesterases, CpFatB4 and CpFatB5, Induce Distinct Gene Expression Reprogramming in Developing Seeds of Brassica napus.

We examined the substrate preference of Cuphea paucipetala acyl-ACP thioesterases, CpFatB4 and CpFatB5, and gene expression changes associated with the modification of lipid composition in the seed, using Brassica napus transgenic plants overexpressing CpFatB4 or CpFatB5 under the control of a seed-specific promoter. CpFatB4 seeds contained a higher level of total saturated fatty acid (FA) content, with 4.3 times increase in 16:0 palmitic acid, whereas CpFatB5 seeds showed approximately 3% accumulation of 10:0 and 12:0 medium-chain FAs, and a small increase in other saturated FAs, resulting in higher levels of total saturated FAs. RNA-Seq analysis using entire developing pods at 8, 25, and 45 days after flowering (DAF) showed up-regulation of genes for ß-ketoacyl-acyl carrier protein synthase I/II, stearoyl-ACP desaturase, oleate desaturase, and linoleate desaturase, which could increase unsaturated FAs and possibly compensate for the increase in 16:0 palmitic acid at 45 DAF in CpFatB4 transgenic plants. In CpFatB5 transgenic plants, many putative chloroplast- or mitochondria-encoded genes were identified as differentially expressed. Our results report comprehensive gene expression changes induced by alterations of seed FA composition and reveal potential targets for further genetic modifications.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

Metabolomic changes in grains of well-watered and drought-stressed transgenic rice.

BACKGROUND: Drought induces a number of physiological and biochemical responses in cereals. This study was designed to examine the metabolite changes in grains of drought-tolerant transgenic rice (Oryza sativa L.) that overexpresses AtCYP78A7 encoding cytochrome P450 protein using proton nuclear magnetic resonance ((1)H-NMR) and gas chromatography/mass spectrometry. RESULTS: Principal component analysis showed that the (1)H-NMR-based profile was clearly separated by soil water status of well-watered and water-deficit. A discrimination of metabolites between transgenic and non-transgenic grains appeared under both watering regimes. Variations in the levels of amino acids and sugars led to the discrimination of metabolites among genotypes. In particular, drought significantly enhanced the levels of γ-aminobutyric acid (GABA, 244.6%), fructose (155.7%), glucose (211.0%), glycerol (57.2%), glycine (65.8%) and aminoethanol (192.4%) in the transgenic grains compared with the non-transgenic control grains. CONCLUSION: These changes in amounts of metabolites may assist in improving drought tolerance in transgenic rice by playing crucial roles in stress-responsive pathways including GABA biosynthesis, sucrose metabolism and antioxidant defenses.

Organization of nif gene cluster in Frankia sp. EuIK1 strain, a symbiont of Elaeagnus umbellata.

The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.

The complete chloroplast genome sequence of a medicinal citrus landrace, Citrus erythrosa Hort. ex Tanaka in Jeju Island, Korea.

Citrus erythrosa (Dongjeongkyool in Korean) is a medicinal citrus landrace that grows in Korea. In this study, we characterized the complete chloroplast (Cp) genome (160,120 bp) of C. erythrosa. The Cp genome was consisted of 4 distinct regions: a large single copy (87,731 bp), a small single copy (18,393 bp), and a pair of inverted repeat regions (26,998 bp). The Cp genome encodes a total of 133 genes including 88 protein-coding genes, 37 tRNA genes and 8 rRNA genes. The phylogenetic analysis reveals that C. erythrosa is a sister group to the clade of species including C. reticulata within the genus Citrus.

APUM23, a nucleolar Puf domain protein, is involved in pre-ribosomal RNA processing and normal growth patterning in Arabidopsis.

Pumilio, an RNA-binding protein that contains tandemly repeated Puf domains, is known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. APUM23 is constitutively expressed, with higher levels in metabolically active tissues, and its expression is up-regulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth, with serrated and scrunched leaves, an abnormal venation pattern, and distorted organization of the palisade parenchyma cells - a phenotype that is reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on this localization, rRNA processing was examined. In apum23, 35S pre-rRNA, and unprocessed 18S and 5.8S poly(A) rRNAs, accumulated without affecting the steady-state levels of mature rRNAs, indicating that APUM23 is involved in the processing and/or degradation of 35S pre-rRNA and rRNA maturation by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 small nucleolar RNAs (snoRNAs) and accumulated RNAs within the nucleolus. Our data suggest that APUM23 plays an important role in plant development via rRNA processing.

Postembryonic seedling lethality in the sterol-deficient Arabidopsis cyp51A2 mutant is partially mediated by the composite action of ethylene and reactive oxygen species.

Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14alpha-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.

Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit (Actinidia deliciosa cv. Hayward).

Genetic transformation using a micro-cross section (MCS) technique was conducted to improve the carotenoid content in kiwifruit (Actinidia deliciosa cv. Hayward). The introduced carotenoid biosynthetic genes include geranylgeranyl diphosphate synthase (GGPS), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), ß-carotene hydroxylase (CHX), and phytoene synthase (PSY). The transformed explants were selected on half-strength MS medium containing 0.001 mg l(-1) of 2,4-D and 0.1 mg l(-1) of zeatin, either 5 mg l(-1) hygromycin or 25 mg l(-1) kanamycin, and 500 mg l(-1) cefotaxime. The genomic PCR, genomic Southern blot analysis, and RT-PCR were performed to confirm the integration and expression of the transgenes. The transformation efficiencies of either kanamycin- or hygromycin-resistant shoots ranged from 2.9 to 22.1% depending on the target genes, and from 2.9 to 24.2% depending on the reporter genes. The selection efficiencies ranged from 66.7 to 100% for the target genes and from 95.8 to 100% for the reporter genes. Changes of carotenoid content in the several PCR-positive plants were determined by UPLC analysis. As a result, transgenic plants expressing either GGPS or PSY increased about 1.2- to 1.3-fold in lutein or ß-carotene content compared to non-transgenic plants. Our results suggest that the Agrobacterium-mediated transformation efficiency of kiwifruit can be greatly increased by this MCS method and that the carotenoid biosynthetic pathway can be modified in kiwifruit by genetic transformation. Our results further suggest that GGPS and PSY genes could be major target genes to increase carotenoid contents in kiwifruit.

Performance of hybrids between abiotic stress-tolerant transgenic rice and its weedy relatives under water-stressed conditions.

Gene transfer from transgenic crops to their weedy relatives may introduce undesired ecological consequences that can increase the fitness and invasiveness of weedy populations. Here, we examined the rate of gene flow from abiotic stress-tolerant transgenic rice that over-express AtCYP78A7, a gene encoding cytochrome P450 protein, to six weedy rice accessions and compared the phenotypic performance and drought tolerance of their hybrids over generations. The rate of transgene flow from AtCYP78A7-overexpressing transgenic to weedy rice varied between 0% and 0.0396%. F1 hybrids containing AtCYP78A7 were significantly taller and heavier, but the percentage of ripened grains, grain numbers and weight per plant were significantly lower than their transgenic and weedy parents. The homozygous and hemizygous F2 progeny showed higher tolerance to drought stress than the nullizygous F2 progeny, as indicated by leaf rolling scores. Shoot growth of nullizygous F3 progeny was significantly greater than weedy rice under water-deficient conditions in a rainout shelter, however, that of homozygous F3 progeny was similar to weedy rice, indicating the cost of continuous expression of transgene. Our findings imply that gene flow from AtCYP78A7-overexpressing transgenic to weedy rice might increase drought tolerance as shown in the pot experiment, however, increased fitness under stressed conditions in the field were not observed for hybrid progeny containing transgenes.

Complete chloroplast genome sequence of a medicinal landrace citrus Jinkyool (Citrus sunki Hort. ex Tanaka) in Jeju Island, Korea.

Citrus sunki (Jinkyool) is a medicinal landrace citrus belonging to the Rutaceae family. We determined the complete chloroplast genome (160,699 bp) of C. sunki CRS0085 in Jeju Island, Korea. The genome is composed of four distinct parts; a large single copy of 87,918 bp, a small single copy of 21,355 bp, and a pair of inverted repeat regions of 25,713 bp. A total of 134 genes including 89 protein-coding genes, 37 tRNA genes, and eight rRNA genes were identified. The phylogenetic tree showed that C. sunki CRS0085 has the closest relationship with C. reticulata within genus Citrus.

Expression of EuNOD-ARP1 encoding auxin-repressed protein homolog is upregulated by auxin and localized to the fixation zone in root nodules of Elaeagnus umbellata.

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.

Expression of a functional type-I chalcone isomerase gene is localized to the infected cells of root nodules of Elaeagnus umbellata.

A putative type-I chalcone isomerase (CHI) cDNA clone EuNOD-CHI was previously isolated from the root nodule of Elaeagnus umbellata [Kim et al. (2003)]. To see if it encodes a functional CHI, we ectopically overexpressed it in the Arabidopsis (Arabidopsis thaliana) transparent testa 5 (tt5) mutant, which is defective in naringenin production and has yellow seeds due to proanthocyanidin deficiency. Ectopic overexpression of EuNOD-CHI resulted in recovery of normal seed coat color. Naringenin produced by CHI from naringenin chalcone was detected in the transgenic lines like in the wild-type, whereas it was absent from the tt5 mutant. We conclude that EuNOD-CHI encodes a functional type-I CHI. In situ hybridization revealed that EuNOD-CHI expression is localized to the infected cells of the fixation zone in root nodules.

Development and characterization of microsatellite primers for Zanthoxylum schinifolium (Rutaceae).

PREMISE OF THE STUDY: Polymorphic microsatellite markers of Zanthoxylum schinifolium (Rutaceae), a promising medicinal plant with effective antibacterial, anticancer, and anti-inflammatory compounds, were developed and evaluated for further genetic studies based on genetic variation among individuals or populations. METHODS AND RESULTS: Following the selective hybridization method, microsatellite-enrichment libraries were constructed. Using these libraries, we obtained 15 polymorphic and three monomorphic microsatellite markers for Z. schinifolium. The number of alleles observed in each of the 15 polymorphic loci ranged from two to eight, and the observed and expected heterozygosities ranged from 0.070 to 0.677 and from 0.093 to 0.688, respectively. Eleven of these developed markers were successfully amplified for Z. piperitum, a related species. CONCLUSIONS: These microsatellite markers can be valuable tools for further genetic studies of Z. schinifolium, such as genetic resource conservation for maintaining breeding material and individual identification for breeding program improvement and variety management.

Development of Genome-Wide SSR Markers from Angelica gigas Nakai Using Next Generation Sequencing.

Angelica gigas Nakai is an important medicinal herb, widely utilized in Asian countries especially in Korea, Japan, and China. Although it is a vital medicinal herb, the lack of sequencing data and efficient molecular markers has limited the application of a genetic approach for horticultural improvements. Simple sequence repeats (SSRs) are universally accepted molecular markers for population structure study. In this study, we found over 130,000 SSRs, ranging from di- to deca-nucleotide motifs, using the genome sequence of Manchu variety (MV) of A. gigas, derived from next generation sequencing (NGS). From the putative SSR regions identified, a total of 16,496 primer sets were successfully designed. Among them, we selected 848 SSR markers that showed polymorphism from in silico analysis and contained tri- to hexa-nucleotide motifs. We tested 36 SSR primer sets for polymorphism in 16 A. gigas accessions. The average polymorphism information content (PIC) was 0.69; the average observed heterozygosity (HO) values, and the expected heterozygosity (HE) values were 0.53 and 0.73, respectively. These newly developed SSR markers would be useful tools for molecular genetics, genotype identification, genetic mapping, molecular breeding, and studying species relationships of the Angelica genus.

Overexpression in Arabidopsis of a plasma membrane-targeting glutamate receptor from small radish increases glutamate-mediated Ca2+ influx and delays fungal infection.

Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.

Differential expression patterns of an acidic chitinase and a basic chitinase in the root nodule of Elaeagnus umbellata.

Two cDNA clones encoding chitinase were isolated from a root nodule cDNA library of Elaeagnus umbellata by the hybridization-competition method. The two clones, EuNOD-CHT1 and EuNOD-CHT2, encode for 335 and 317 amino acid residues with the molecular mass of mature proteins being 33.3 and 31.1 kDa, respectively. The two chitinases showed similar protein structures consisting of four domains: hydrophobic signal peptide domain, cysteine-rich chitin-binding domain, hinge domain, and catalytic domain. The EuNOD-CHT1 gene showed similar expression levels in root nodules and leaves, with no detection of transcripts in the roots. The EuNOD-CHT2 gene was expressed at similarly high levels in the roots and root nodules, but at a very low level in the leaves. In situ hybridization showed that EuNOD-CHT1 transcripts were strongly detected in the meristem zone, but weakly detected in the outer cortex layer of the root nodule and in the uninfected cells of the fixation zone. On the other hand, EuNOD-CHT2 transcripts were strongly detected in the infected cells of the fixation zone and central vascular system, but weakly detected in the senescence zone. Our results suggest that the two chitinases may play different biological roles in the root nodule. EuNOD-CHT2 may be involved in a defense response against internal symbionts, external pathogens, or both, while EuNOD-CHT1 may be involved in normal plant development as well as in a defensive role against external pathogens.

Molecular cloning and complementation analysis of nifV gene from Frankia EuIK1 strain.

The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function. The nifV ORF consists of 1245 bp, which encodes 414 amino acids. However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF. The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp. FaC1. In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found. The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region. In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization. A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV. The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K. pneumoniae mutant, respectively. These results suggested that Frankia EuIK1 nifV is a functional gene.

Analysis of the root nodule-enhanced transcriptome in soybean.

For high throughput screening of root nodule-enhanced genes, cDNA libraries specific for three different developmental stages of soybean root nodules were constructed after inoculation with Bradyrhizobium japonicum USDA110. 5,469 cDNA clones were sequenced and grouped into 2,511 non-redundant (nr) ESTs consisting of 769 contigs and 1,742 singletons. Using similarity searches against several public databases we constructed a functional classification of the ESTs into root nodule-specific nodulin genes, stress-responsive genes and genes related to carbon and nitrogen metabolism. We also constructed a cDNA microarray with 382 selected clones that appeared to be up-regulated in the root nodule. Using the microarray we compared the transcript levels of uninfected roots and root nodules from four developmental stages. We identified 81 genes that were differentially expressed, and grouped them into seven clusters according to the similarity of their expression profiles, using a hierarchical clustering algorithm. Clusters 1, 2, 3, and 6, comprised of 58 genes, showed root nodule-enhanced expression. The information from this study will be used to analyze the roles of root nodule-specific genes and signaling pathways during root nodule development.

Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions.