Reproductive barriers are often observed in progenies derived from crosses between relatively distant species, such as cultivated varieties and their wild relatives. Such barriers hinder the transfer of agronomically important genes from one species to another. In the process of constructing a series of introgression lines (ILs) between Oryza sativa japonica cv. Koshihikari and wild species O. nivara, we observed the occurrence of hybrid breakdown, a reproductive barrier observed in F2 and later generations. Plants affected by this hybrid breakdown show extremely weak growth and die before heading. Linkage analysis and construction of ILs indicated that the locus -hbd1(t), located on the short arm of chromosome 2 of O. nivara, induces hybrid breakdown in the Koshihikari genetic background. hbd1(t) is identical to hbd1, which was previously identified in a cross between Koshihikari and Nona Bokra (indica) cultivars of O. sativa. We present genetic evidence that hbd1(t) regulates hybrid breakdown and its location within 50 kb. Possible reasons for this locus being responsible for hybrid breakdown in inter- and intraspecific crosses are discussed.
Radish (Raphanus sativus L.) is a useful vegetable with diverse features and worldwide distribution; however, the diversification and domestication history of cultivated radish has not been well documented. In order to understand genetic relationships among radishes around the world and the resulting diversity, we analyzed 65 accessions of cultivated radish collected from 21 Eurasian and North African countries using 221 amplified fragment-length polymorphism (AFLP) markers. These accessions formed four groups according to their provenance (Europe, Middle East, South Asia, and East Asia) in a neighbor-joining (NJ) tree. Despite geographical barriers, there might thus be a frequent exchange of germplasms within each region. The average genetic diversity did not differ significantly among the groups, ranging from 0.267 (Middle East) to 0.297 (East Asia), indicating that no obvious bottleneck effect in each region has occurred during the spread of this species.
In rice (Oryza sativa L.) plants, accumulation and remobilization of nitrogen (N) are essential physiological processes that determine grain yield and quality. The objectives of the present study were to identify and characterize the genomic regions associated with this N dynamics by quantitative trait locus (QTL) analysis. In the present study, 191 recombinant inbred lines (F7) derived from a cross between Milyang 23 (Indica-type) and Akihikari (Japonica-type) were repeatedly evaluated for leaf nitrogen (N) concentration throughout four cropping seasons in Joetsu, Japan and Los Baños, Philippines, to perform interval mappings using a 182 RFLP marker-based linkage map. The locations and effects of the QTLs detected showed that the N dynamics was controlled by 15 genomic regions classifed into three groups. Four regions in Group I (chromosomes 1, 2, 7 and 8) affected only the N concentration before heading, i.e., N accumulation during the vegetative phase; whereas eight regions in Group II (chromosomes 2, 3, 4, 6, 7, 9, 10 and 12) affected the N concentration after heading, i.e., N remobilization during the reproductive phase. Only three regions (chromosomes 1, 2 and 10) in Group III exerted effects on both N concentrations in the vegetative and reproductive phases. In 12 of these 15 regions, expression of the QTLs depended on the cropping seasons and sites. Statistical tests enabled to detect significant genotype × stage and genotype × season interaction effects for nine and eight regions, respectively. Characterization of the 15 regions demonstrated that the genetic control of N dynamics involves a number of genes whose effects were enhanced or suppressed by plant ontogeny and the growth environment.
We isolated transformation-competent artificial chromosome (TAC) clones harboring a high-molecular-weight glutenin subunit (HMW-GS) gene and a low-molecular-weight GS (LMW-GS) gene from wheat genomic DNA, and developed transgenic rice lines with these genes using a transformation system mediated by Agrobacterium tumefaciens. We developed two lines of transgenic rice, one expressing the gene coding for HMW-GS and the other expressing the gene coding for LMW-GS. By crossing these transgenic lines, a novel line harboring both genes was developed and the expression of GS proteins was analyzed. This is the first study indicating that the two kinds of wheat GSs, HMW-GS and LMW-GS, accumulated in rice endosperm. In all the transgenic lines, the introduced GS genes were expressed in the rice endosperm, and the expressed proteins were processed at the same site as the mature GS protein in wheat seeds, forming insoluble polymeric proteins similar to those found in wheat. It was suggested that the protein-processing system was conserved between rice and wheat, and that it was possible to produce wheat gluten using the protein-processing system of rice.
Callus growth and shoot regeneration were examined in cultures of immature barley embryos incubated under various combinations of a week under a 16-h photoperiod and a week under continuous darkness. Incubation in darkness for four weeks, during which calli were formed, enhanced shoot regeneration in ‘K-3’ and ‘Kanto Nijo-5’, but inhibited it in ‘Lenins’. ‘K-3’ and ‘Kanto Nijo-5’, incubated in darkness during the first two weeks of callus induction, followed by two weeks of a 16-h photoperiod (D2L2), showed a higher percentage of shoot regeneration than those incubated for two weeks with a 16-h photoperiod followed by two weeks of darkness (L2D2). Nevertheless, when the light conditions, D2L2 and L2D2, were combined into the same periods of a 16-h photoperiod and continuous darkness, except for the order, light conditions affected shoot regeneration differently. The early stage of callus induction seems to be sensitive to light. The expression ratios of the auxin-responsive gene (AUX/IAA) to the cytokinin-responsive gene (WPK4) were increased in continuous darkness in ‘K-3’ and ‘Kanto Nijo-5’. The effects of light qualities (white, red, far-red, and blue) on callus growth and shoot regeneration were also examined. Blue light inhibited shoot regeneration, as did white light, in ‘K-3’ and ‘Kanto Nijo-5’, and far-red light in ‘Kanto Nijo-5’. Light probably controls shoot regeneration from calli by modifying cytokinin levels and/or response; blue light signals act in photo-inhibition of shoot regeneration in immature barley embryo culture.
Pepper (Capsicum annuum L.) anthracnose, caused by Colletotrichum spp., is an important disease in many Asian countries. Recently, it was found that a local Korean variety, C. annuum ‘Daepoong-cho’, had resistance to C. capsici. Inheritance of resistance to C. capsici was analyzed in segregating populations derived from a cross of ‘Yeoju’ × ‘Daepoong-cho’. An allelism test was carried out in F1 and F2 progenies derived from the cross of ‘Daepoong-cho’ × ‘AR’. ‘AR’ is an anthracnose-resistant breeding line derived from C. chinense Jacq. ‘PBC 932’. Detached mature green fruits were inoculated using the microinjection method. Disease response was evaluated using disease incidence, overall lesion diameter, and true lesion diameter at 7 days after inoculation. The segregation ratios of resistance and susceptibility to C. capsici in F2 and BCR populations derived from the cross of ‘Yeoju’ × ‘Daepoong-cho’ were fitted to the 1 : 3 and 1 : 1 Mendelian model, respectively. This indicates that the resistance of ‘Daepoong-cho’ to C. capsici is controlled by a single recessive gene. The results of the allelism test indicated that the two resistant lines, ‘Daepoong-cho’ and ‘AR’, possessed the same resistance gene to C. capsici, even though the resistance genes of these two lines originated from different Capsicum species, C. annuum and C. chinense.
Rutin, anthocyanins and proanthocyanidins are representative flavonoids that are synthesized through the flavonoid biosynthetic pathway in common buckwheat (Fagopyrum esculentum). These flavonoids have beneficial properties such as antioxidant activity. The basal buckwheat stem is usually red or pink owing to an accumulation of anthocyanins. We found novel spontaneous green stem (GS) mutants in a common buckwheat population. To characterize GS mutants physiologically and genetically, and to deduce where the flavonoid biosynthetic pathway was disrupted and whether other flavonoids were affected by this disruption of anthocyanin synthesis, we measured the anthocyanin, rutin and proanthocyanidin contents in seedlings and performed genetic analysis. HPLC analysis of GS mutants showed no anthocyanin production, but rutin and proanthocyanidins accumulated in the seedlings. GS plants showed no red pigmentation in stems, petioles, leaf veins and anthers. Genetic analysis indicated that GS mutation involves a single recessive gene (designated tentatively as gs1). The proanthocyanidin content of GS plants in the F2 segregating population was significantly higher than in the wild type. The GS phenotype might be caused by the lack of an enzyme or by a defect of transcriptional factor between leucocyanidin or cyanidin and anthocyanin in the biosynthetic pathway.
Barley includes semi-dwarf varieties, called uzu, which are localized in parts of southwestern Japan, the southern Korea peninsula, and coastal areas of China. The uzu phenotype possesses dark green leaves and short coleoptiles, awns, and panicles. It is controlled by a single recessive gene: uzu. Uzu results from a mutation in the brassinosteroid receptor kinase gene (HvBRI1). Brassinosteroid synergistically acts with auxin on plant morphology, which is an important plant hormone for tissue culture. For this study, tissue culture traits, including callus growth and shoot regeneration capability, were examined in F2 populations derived from crosses between normal and uzu lines, and in isogenic lines for the uzu gene. The uzu genotype shows a lower percentage of shoot regeneration than the normal genotype in F2 populations and isogenic lines. The uzu gene negatively affects shoot regeneration. No significant differences were found in callus growth capability between uzu and normal genotypes. Uzu isogenic lines show higher sensitivity to exogenous auxin for callus initiation than normal lines, when immature embryos were incubated on media supplemented with several concentrations of 2,4-D under culture at higher temperature (25°C). Tissue culture traits of uzu might be regulated through cross-talk between brassinosteroid and auxin.
To apply genomic information of the model legume Lotus japonicus to soybean, the characteristics of the soybean genome in reference to the genome of L. japonicus were investigated. Macrosynteny between soybean and L. japonicus was analyzed by mapping the same cDNA clones on the maps of both species by the RFLP method, and by identifying the positions of orthologs on the L. japonicus map for cDNA markers located on the soybean map. Relatively large synteny blocks were observed between a few linkage groups of L. japonicus and soybean. The major parts of the soybean linkage groups consisted of mosaics of smaller segments syntenic with the L. japonicus genome. The presence of many homoeologous regions on different soybean linkage groups was suggested from the distribution of paralogs and orthologs. To investigate the microsynteny between soybean and L. japonicus, three soybean BAC clones were selected for the GmNFR1a, GmNFR1b and Nts1 genes mapped on the macrosyntenic regions of the linkage groups D1b, B2 and H, respectively. We revealed a significantly high level of collinearity between these BAC clones and corresponding homologous genomic regions of L. japonicus. The information of L. japonicus could be used for the development of DNA markers, map-based cloning and assembling process of genome sequencing in soybean.
Deterioration of rice grains and the development of stale flavor during storage are serious problems that can reduce the quality of stored rice. Lipoxygenase (LOX) catalyzes the peroxidation of lipids in rice grains, leading to the formation of volatile compounds. LOX activity in rice seeds is localized in the bran fraction, and LOX-3 is the major isozyme component. We previously found that the Thai variety “Daw Dam” lacks LOX-3 protein and that LOX-3 null rice has less stale flavor during storage than normal LOX-3 rice. Thus, introduction of the LOX-3 null phenotype in mature seeds would facilitate the retention of high quality during storage. Although the LOX-3 null phenotype is inherited as a single recessive trait, the widely used screening method for LOX-3 null strains involves Western blotting with a progeny test that requires considerable time and effort. To develop a convenient LOX-3 null strain detection method, we performed linkage analysis between the LOX-3 null phenotype and restriction fragment length polymorphism and simple sequence repeat markers using F2 progeny from “Daw Dam” and “Nipponbare”, and mapped the LOX-3 null phenotype between RM6736 and RM6329 on chromosome 3 containing three putative LOX genes. LOX-3 protein was purified using an affinity column bound with anti-LOX-3 monoclonal antibodies, and its amino acid sequences were determined. These sequences matched only those of the protein encoded by Os03g0700400, one of the three putative LOX genes. In DNA sequencing analysis, the “Daw Dam” allele of Os03g0700400 showed transition mutation from G to A on the seventh exon. This change resulted in a stop codon, implying a nonsense mutation in the “Daw Dam” allele. Based on these results, Os03g0700400 was confirmed to be the rice seed LOX-3 gene. Using these DNA sequence data, we developed a cleaved amplified polymorphic sequence and dot-blot-single nucleotide polymorphism analyses for screening and breeding LOX-3 null rice varieties. These methods are easy and cost-effective for screening LOX-3 null strains.
In radish, the mitochondrial gene orf138 found in Ogura cytoplasm is responsible for male sterility, whereas the nuclear restorer gene (Rf gene) inhibits the expression of orf138. To investigate the distribution of the Rf gene for Ogura male sterile cytoplasm in Japanese wild radish, 226 plants collected from 15 regions in Japan and two additional sites of Korea were used as materials. PCR analysis indicated that 42% of the wild radishes possessed orf138. On the other hand, 207 plants (91.6%) were judged to have an Rf gene by the observation of pollen fertility in wild radishes themselves and in hybrids obtained by crosses with a male sterile variety having Ogura cytoplasm. For plants having restorer function, we analyzed the nucleotide sequence of the Rf gene, orf687, identified to date. Among the 207 plants having the Rf gene, 199 amplified the orf687 fragment of the expected size by PCR. PCR products were further analyzed by mismatch-specific endonuclease digestion and PCR-RFLP analysis. As a result, plants that showed an identical RFLP pattern to the known Rf type of orf687 were restricted to 30 plants (14.5% of the 207 plants) collected mainly in populations of southern region of Japan. It was also found that most of the plants (148 plants; 71.5% of 207 plants) possessed the sequence of the orf687 corresponding to the rf genotype lacking the restorer function. We conclude that the Rf gene widely distributed in Japanese wild radish is not orf687.