High-Density Linkage Maps from Japanese Rice japonica Recombinant Inbred Lines Using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di).

The genetic dissection of agronomically important traits in closely related Japanese rice cultivars is still in its infancy mainly because of the narrow genetic diversity within japonica rice cultivars. In an attempt to unveil potential polymorphism between closely related Japanese rice cultivars, we used a next-generation-sequencing-based genotyping method: genotyping by random amplicon sequencing-direct (GRAS-Di) to develop genetic linkage maps. In this study, four recombinant inbred line (RIL) populations and their parents were used. A final RIL number of 190 for RIL71, 96 for RIL98, 95 for RIL16, and 94 for RIL91 derived from crosses between a common leading Japanese rice cultivar Koshihikari and Yamadanishiki, Taichung 65, Fujisaka 5, and Futaba, respectively, and the parent plants were subjected to GRAS-Di library construction and sequencing. Approximately 438.7 Mbp, 440 Mbp, 403.1 Mbp, and 392 Mbp called bases covering 97.5%, 97.3%, 98.3%, and 96.1%, respectively, of the estimated rice genome sequence at average depth of 1× were generated. Analysis of genotypic data identified 1050, 1285, 1708, and 1704 markers for each of the above RIL populations, respectively. Markers generated by GRAS-Di were organized into linkage maps and compared with those generated by GoldenGate SNP assay of the same RIL populations; the average genetic distance between markers showed a clear decrease in the four RIL populations when we integrated markers of both linkage maps. Genetic studies using these markers successfully localized five QTLs associated with heading date on chromosomes 3, 6, and 7 and which previously were identified as Hd1, Hd2, Hd6, Hd16, and Hd17. Therefore, GRAS-Di technology provided a low cost and efficient genotyping to overcome the narrow genetic diversity in closely related Japanese rice cultivars and enabled us to generate a high density linkage map in this germplasm.

Apple latent spherical virus vector-induced flowering for shortening the juvenile phase in Japanese gentian and lisianthus plants.

MAIN CONCLUSION: Infection by apple latent spherical virus (ALSV) vectors that promote the expression of Arabidopsis thaliana FLOWERING LOCUS T ( AtFT ) or Gentiana triflora GtFT s accelerates flowering in gentian and lisianthus plants. Apple latent spherical virus (ALSV) has isometric virus particles (25 nm in diameter) that contain two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). ALSV vectors are used for foreign gene expression and virus-induced gene silencing in a broad range of plant species. Here, we report the infection by ALSV vectors that express FLOWERING LOCUS T (AtFT) from Arabidopsis thaliana or its homolog GtFT1 from Gentiana triflora in three gentian cultivars ('Iwate Yume Aoi' [early flowering], 'Iwate' [medium flowering], and 'Alta' [late flowering]), and two lisianthus cultivars ('Newlination Pink ver. 2' and 'Torukogikyou daburu mikkusu') promotes flowering within 90 days post-inoculation using particle bombardment. Additionally, seedlings from the progeny of the early-flowering plants were tested by tissue blot hybridization, and the results showed that ALSV was not transmitted to the next generation. The promotion of flowering in the family Gentianaceae by ALSV vectors shortened the juvenile phase from 1-3 years to 3-5 months, and thus, it could be considered as a new plant breeding technique in ornamental gentian and lisianthus plants.

The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response.

Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2 Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G-A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants.

MutMap+: genetic mapping and mutant identification without crossing in rice.

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.

Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs, causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.).

The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice.

Possible role of early flowering 3 (ELF3) in clock-dependent floral regulation by short vegetative phase (SVP) in Arabidopsis thaliana.

Circadian clock proteins play key roles in adaptations of plants to diurnal environmental conditions. The photoperiodic flowering response is one of the mechanisms of adaptation to seasonal changes in the lengths of day and night. Double mutations in two clock genes, late elongated hypocotyl (LHY) and circadian clock associated 1 (CCA1), accelerated flowering under short days (SDs) but delayed flowering under continuous light (LL) in Arabidopsis thaliana. The mechanism underlying the late flowering of lhy;cca1 mutants under LL was investigated here. Late flowering of plants with overexpression of short vegetative phase (SVP) was much more pronounced under SDs and enhanced by constans 2 (co-2) under long days (LDs), suggesting that SVP and CO act independently in the photoperiodic flowering pathway. However, how SVP and flowering locus C (FLC) mediated the effects of LHY/CCA1 and thus influenced flowering time was not completely clear. A mutant line lhy;cca1 in the Landsberg erecta (Ler) background was established, ethyl methanesulfonate (EMS)-mutagenized and used to screen suppressors of late flowering of lhy;cca1 under LL. Mutations in the clock gene early flowering 3 (ELF3) were identified as suppressors. Overexpression and loss-of-function of ELF3 influenced SVP protein accumulation. Therefore, we propose that, as well as the classical GIGANTEA (GI)-CO pathway, LHY/CCA1 regulates a pathway negatively controlling flowering locus T (FT), possibly via ELF3-SVP/FLC.