Characterization of a New Citrus Mutant Induced by Gamma Irradiation with a Unique Fruit Shape, Gwonje-Early, and Determination of Specific Selection Markers Using Allele-Specific PCR.

Gamma-ray irradiation is one of the most widely used mutagens worldwide. We previously conducted mutation breeding using gamma irradiation to develop new Citrus unshiu varieties. Among these mutants, Gwonje-early had an ovate shape, a protrusion of the upper part of the fruit, and a large fruit size compared with wild-type (WT) fruits. We investigated the external/internal morphological characteristics and fruit sugar/acid content of Gwonje-early. Additionally, we investigated genome-wide single-nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) variants in Gwonje-early using whole-genome re-sequencing. Functional annotation by Gene Ontology analysis confirmed that InDels were more commonly annotated than SNPs. To identify specific molecular markers for Gwonje-early, allele-specific PCR was performed using homozygous SNPs detected via Gwonje-early genome re-sequencing. The GJ-SNP1 and GJ-SNP4 primer sets were effectively able to distinguish Gwonje-early from the WT and other commercial citrus varieties, demonstrating their use as specific molecular markers for Gwonje-early. These findings also have important implications in terms of intellectual property rights and the variety protection of Gwonje-early. Our results may provide insights into the understanding of morphological traits and the molecular breeding mechanisms of citrus species.

Identification of Late Ripening Citrus Mutant, Ara-unshiu (Citrus unshiu), and Its Selectable Marker.

'Miyagawa-wase' mandarin (Citrus unshiu Marc. cv. Miyagawa-wase early) is one of the most widely cultivated citrus varieties on Jeju Island in Korea. Mutation breeding is a useful tool for the induction of genetic diversity for the rapid creation of new plant variants. We previously reported the use of gamma irradiation for the development of new citrus varieties. Here, we report a new mutant, Ara-unshiu, with a unique late fruit ripening phenotype. We investigated the fruit morphological characteristics including weight, vertical/transverse diameter, peel thickness, hardness, and color difference, as well as sugar and acid contents of the Ara-unshiu compared to wild-type controls. We then used whole genome re-sequencing and functional annotation by gene ontology to identify and characterize single nucleotide polymorphism (SNP) and insertion/deletion (InDel) variants in the Ara-unshiu, finding a greater abundance of annotated genes containing InDels compared to SNPs. Finally, we used allele-specific PCR to identify molecular markers among the homozygous SNPs detected from the Ara-unshiu genome sequencing. We report a primer set that effectively distinguishes the Ara-unshiu from the wild-type control and other citrus varieties. Our findings provide insights into the mechanisms controlling the timing of fruit ripening and tools for the molecular breeding of citrus varieties.

The Citrus Mutant Jedae-unshiu Induced by Gamma Irradiation Exhibits a Unique Fruit Shape and Increased Flavonoid Content.

Satsuma mandarin (Citrus unshiu Marc. cv. Miyagawa-wase) is the most widely cultivated citrus variety in Korea. Although most varieties are imported from Japan, efforts have focused on developing new domestic varieties. We produced mutants by irradiating C. unshiu Marc. cv. Miyagawa-wase scions with gamma rays and grafting them onto C. unshiu Marc. cv. Miyagawa-wase branches. We compared the characteristics of these mutants with Miyagawa-wase fruit as a control. A mutant line named Jedae-unshiu with a unique fruit shape was selected for investigation in detail. The phenotype of Jedae-unshiu fruit demonstrated vertical troughs on the flavedo, smooth albedo without rough protruding fibers, and good adhesion between peel and flesh. In addition, Jedae-unshiu had thicker peels and higher fruit hardness than the control. Higher levels of hesperetin and narirutin, representative flavonoids, accumulated in the peel and flesh of Jedae-unshiu than those of the control. Cellular-level microscopic observations of the mature fruit peels demonstrated epidermal cell disruption in the control but not in Jedae-unshiu. Our results suggest that Jedae-unshiu has high possibility for development as a good storage variety containing large amounts of flavonoids, in addition to potential for ornamental value due to the unique shape of the fruit.

Transgenerational activation of an autonomous DNA transposon, Dart1-24, by 5-azaC treatment in rice.

KEY MESSAGE: Dart1-24, one of the 37 autonomous DNA transposon Dart1s, was heritably activated by the demethylation of the 5' region following 5-azaC treatment of rice seeds. Transposons are controlled by epigenetic regulations. To obtain newly activated autonomous elements of Dart1, a DNA transposon, in rice, seeds of a stable pale yellow leaf (pyl-stb) mutant caused by the insertion of nDart1-0, a nonautonomous element in OsClpP5, were treated with 5-azaC, a demethylating agent. In the 5-azaC-treated M1 plants, 60-70% of the plants displayed variegated pale yellow leaf (pyl-v) phenotype, depending on the concentration of 5-azaC used, suggesting that inactivated Dart1 might become highly activated by 5-azaC treatment and nDart1-0 was excised from OsClpP5 by the activated Dart1s. Although the M2 plants derived from most of these pyl-v plants showed stable pyl phenotypes, some variegated M1 plants generated pyl-v M2 progeny. These results indicated that most M1 pyl-v phenotypes at M1 were not heritable. Dart1-24, 1-27 and 1-28 were expressed in the M2 pyl-v plants, and mapping analysis confirmed that Dart1-24 was newly activated. Further, the transgenerational activation of Dart1-24 was demonstrated to be caused by the demethylation of nucleotides in its 5' region.

Disposable all-printed electronic biosensor for instantaneous detection and classification of pathogens.

A novel disposable all-printed electronic biosensor is proposed for a fast detection and classification of bacteria. This biosensor is applied to classify three types of popular pathogens: Salmonella typhimurium, and the Escherichia coli strains JM109 and DH5-α. The proposed sensor consists of inter-digital silver electrodes fabricated through an inkjet material printer and silver nanowires uniformly decorated on the electrodes through the electrohydrodynamic technique on a polyamide based polyethylene terephthalate substrate. The best sensitivity of the proposed sensor is achieved at 200 µm teeth spaces of the inter-digital electrodes along the density of the silver nanowires at 30 × 103/mm2. The biosensor operates on ±2.5 V and gives the impedance value against each bacteria type in 8 min after sample injection. The sample data are measured through an impedance analyzer and analyzed through pattern recognition methods such as linear discriminate analysis, maximum likelihood, and back propagation artificial neural network to classify each type of bacteria. A perfect classification and cross-validation is achieved by using the unique fingerprints extracted from the proposed biosensor through all the applied classifiers. The overall experimental results demonstrate that the proposed disposable all-printed biosensor is applicable for the rapid detection and classification of pathogens.

Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)- and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.

Activation and epigenetic regulation of DNA transposon nDart1 in rice.

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.

Targeted disruption of an orthologue of DOMAINS REARRANGED METHYLASE 2, OsDRM2, impairs the growth of rice plants by abnormal DNA methylation.

Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages.

AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Genetic evidence that DNA methyltransferase DRM2 has a direct catalytic role in RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic modification in plants. We report here the identification of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) in a forward screen for mutants defective in RdDM in Arabidopsis thaliana. The finding of a mutation in the presumptive active site argues in favor of direct catalytic activity for DRM2.

Characterization of autonomous Dart1 transposons belonging to the hAT superfamily in rice.

An endogenous 0.6-kb rice DNA transposon, nDart1-0, was found as an active nonautonomous element in a mutable virescent line, pyl-v, displaying leaf variegations. Here, we demonstrated that the active autonomous element aDart in pyl-v corresponds to Dart1-27 on chromosome 6 in Nipponbare, which carries no active aDart elements, and that aDart and Dart1-27 are identical in their sequences and chromosomal locations, indicating that Dart1-27 is epigenetically silenced in Nipponbare. The identification of aDart in pyl-v was first performed by map-based cloning and by detection of the accumulated transposase transcripts. Subsequently, various transposition activities of the cloned Dart1-27 element from Nipponbare were demonstrated in Arabidopsis. Dart1-27 in Arabidopsis was able to excise nDart1-0 and Dart1-27 from cloned sites, generating footprints, and to integrate into new sites, generating 8-bp target site duplications. In addition to Dart1-27, Nipponbare contains 37 putative autonomous Dart1 elements because their putative transposase genes carry no apparent nonsense or frameshift mutations. Of these, at least four elements were shown to become active aDart elements in transgenic Arabidopsis plants, even though considerable sequence divergence arose among their transposases. Thus, these four Dart1 elements and Dart1-27 in Nipponbare must be potential autonomous elements silenced epigenetically. The regulatory and evolutionary implications of the autonomous Dart1 elements and the development of an efficient transposon-tagging system in rice are discussed.

An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice.

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.