Tetsuya Nakazaki

Mobilization of a transposon in the rice genome.

Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence, a useful model for analysing the genome structure of grasses. To practice efficient rice breeding through genetic engineering techniques, it is important to identify the economically important genes in this crop. The use of mobile transposons as gene tags in intact plants is a powerful tool for functional analysis because transposon insertions often inactivate genes. Here we identify an active rice transposon named miniature Ping (mPing) through analysis of the mutability of a slender mutation of the glume—the seed structure that encloses and determines the shape of the grain. The mPing transposon is inserted in the slender glume (slg) mutant allele but not in the wild-type allele. Search of the O. sativa variety Nipponbare genome identified 34 sequences with high nucleotide similarity to mPing, indicating that mPing constitutes a family of transposon elements. Excision of mPing from slg plants results in reversion to a wild-type phenotype. The mobility of the transposon mPing in intact rice plants represents a useful alternative tool for the functional analysis of rice genes.

High Potential of a Transposon mPing as a Marker System in japonica × japonica Cross in Rice

Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare × Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). In addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare × Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.

QTL analysis of seed-flooding tolerance in soybean (Glycine max [L.] Merr.)

In soybean (Glycine max [L.] Merr.), varieties with seed-flooding tolerance at the geminating stage are desirable for breeding in countries with much rainfall at sowing time. Our study revealed great intervarietal variation in seed-flooding tolerance as evaluated by germination rate (GR) and normal seedling rate (NS). Pigmented seed coat and small seed weight tended to give a positive effect on seed-flooding tolerance. Subsequently, QTL analysis of GR and NS were performed and a total of four QTLs were detected. Among them, Sft1 on the linkage group H (LG_H) exhibited a large effect on GR after a 24-h treatment; however, Sft2 near the I locus on LG_A2 involved in seed coat pigmentation exhibited the largest effect on seed-flooding tolerance. Sft1, Sft3 and Sft4 were independent of seed coat color and seed weight. Based on the results, we discussed the physiological effects of genetic factors responsible for seed-flooding tolerance in soybean.

Production of doubled-haploid plants from Lilium longiflorum Thunb. anther culture

The effects of different media, light conditions, and pretreatments by cold, and by centrifugation were evaluated on Lilium longiflorum Thunb. anther culture. A high percentage of responding anthers was achieved with N6 medium, by cold pretreatment and under dark conditions. Callus induction was promoted under darkness, but was inhibited by light. Anthers from flower buds between 30 and 46 mm in length containing early- and mid-uninucleated microspores produced callus. A total of 132 plantlets with bulblet and leaves were regenerated. Among anther-callus regenerated plants no albino or morphological variant was observed. The presence of both haploid and diploid cells in the same root tip of regenerated plants indicated that chromosome doubling may have occurred spontaneously. Microscopic observation of microspores from cultured anthers showed the presence of multicellular grains at various stages. These observations suggested that microspore-derived plants were produced by the androgenesis process. This fact was confirmed by isozyme analysis. The potential use of doubled-haploid (DH) plants from anther culture in relation to cultivar improvement in L. longiflorum Thunb. is discussed.

Efficient callus induction and plant regeneration from filaments with anther in lily (Lilium longiflorum Thunb.)

Abstract Bulb scale propagation makes it difficult to obtain a large number of bulblets from disease-free stocks in a short time. The establishment of improved micropropagation procedures by in vitro culture is therefore desirable. Easter lily (Lilium longiflorum Thunb.) filaments with and without anther were excised and cultured in vitro with different media and culture conditions. In cultures of filaments with anther, callus developed and led to bulb, shoot, and root formation, whereas in cultures of filaments lacking anther, callus development did not occur. Among the various media tested, the B5 medium combined with darkness and the N6 medium combined with darkness or light, both supplemented with 9% sucrose, proved to be superior. A total of 1260 plants were regenerated from callus, acclimatized under a mist, and transferred to the greenhouse with a 100% success rate. No morphological abnormalities were observed among plants regenerated from filament-derived callus and all plants displayed isozyme banding patterns identical to the original cultivar. Chromosome observations revealed that all callus-regenerated plantlets tested were diploid (2n=24). The results suggest that in vitro culture of filaments with anther can be cultured for mass propagation.

Detection of a 4DL chromosome segment translocated to rye chromosome 5R in an advanced hexaploid triticale line Bronco 90

Bronco 90 is an advanced line of hexaploid triticale and was reported to be a 2D(2R) chromosome substitution type. In F1 hybrids of this triticale with bread wheat, however, a meiotic configuration of 16 bivalents and 10 univalents was frequently observed indicating the presence of an additional D(R) chromosome substitution or D/R translocation. To determine the chromosome constitution of Bronco 90, C-banding and fluorescent in situ hybridization techniques were applied to somatic and meiotic metaphase chromosomes. These analyses revealed that in Bronco 90, the terminal 7% of the long arm of rye chromosome 5R is derived from the long arm of chromosome 4D. This translocated chromosome (5RS.5RL-4DL) and telosome 4DL formed metaphase I bonds at a frequency of 71%, demonstrating the significance of small terminal chromosome segments for pairing. This novel rye-wheat translocation is probably generated by homoeologous crossing-over because the distal region of 5RL is known to be homoeologous to that of 4DL. Possible association of this translocation with the absence of hairy peduncle character in Bronco 90 is discussed.

Wide genetic variation in phenolic compound content of seed coats among black soybean cultivars.

Black soybeans have been used as a food source and also in traditional medicine because their seed coats contain natural phenolic compounds such as proanthocyanidin and anthocyanin. The objective of this research is to reveal the genetic variation in the phenolic compound contents (PCCs) of seed coats in 227 black soybean cultivars, most of which were Japanese landraces and cultivars. Total phenolics were extracted from seed coats using an acidic acetone reagent and the proanthocyanidin content, monomeric anthocyanin content, total flavonoids content, total phenolics content, and radical scavenging activity were measured. The cultivars showed wide genetic variation in PCCs. Each of the contents was highly correlated with one another, and was closely associated with radical scavenging activity. PCCs were also moderately associated by flowering date but not associated by seed weight. Cultivars with purple flowers had a tendency to produce higher PCCs compared with cultivars with white flowers, suggesting that the W1 locus for flower color can affect phenolic compound composition and content. Our results suggest that developing black soybean cultivars with high functional phenolic compounds activity is feasible.

Stability Verification of the Effects of Stem Determination and Earliness of Flowering on Green Stem Disorder of Soybean against Genetic Background and Environment

Abstract Green stem disorder (GSD) of soybean reduces harvesting efficiency and negatively impacts seed appearance when mechanical harvesting is employed. Two recombinant inbred populations were investigated for the effects of segregating stem determination and flowering time on GSD at two different locations, Kyoto and Akita, over two years. Although the severity of GSD of each line varied considerably with the location, the scores showed significant correlation with the environment. Quantitative trait locus (QTL) analysis revealed a strong and consistent QTL for GSD severity in one population of recombinantinbred lines (RILs) across the environments at the Dt1 locus, which governs stem growth habits, and the determinate growth genotypes showed evident symptoms of GSD. However, QTLs were not detected near the Dt1 locus in the other population. Thus, it was unclear if the responsible gene was identical to the stem determination gene. The early flowering genotype showed a more severe symptom of GSD in both populations, but this effect was dependent on theallele at the Dt1 locus. The effect of another QTL detected in the latter population also depended on theallele at the Dt1 locus. Our results indicated that the genetic factor at the Dt1 locusand the factor controlling flowering time influenced the severity of GSD at each location and year and that their effects and interaction complicated the genetic control of the occurrence of GSD.

Active Transposons in Rice

The first ‘transposon’ – that of the ‘controlling element’ of maize – was discovered by Mclintock more than half a century ago. This element was detected as a factor controlling the mutable character of kernel pigmentation. In higher plants, many mutable traits have bee found, especially for genes involved in pigmentation and endosperm quality. Recent molecular analyses have revealed that many of these mutable traits are controlled by transposons (Fedoroff et al. 1983, 1984; Bonas et al. 1984; Brown et al. 1989; Inagaki et al. 1994). Transposons or transposable elements were originally defined as mobile genetic elements; that is, as DNA fragments with the ability to move to new chromosomal locations (this is known as transposition). With advances in the knowledge of the sequence structures of transposons, it has become clear that they include a number of DNA elements which had probably lost mobility because of mutations on the sequences or other factors. Transposable elements are divided into two groups according to the mode of propagation: retrotransposons (class I elements) and DNA transposable elements (transposons in a narrow sense, class II). While the former moves through RNA intermediate by the action of reverse transcriptase, the latter moves in a DNA form through a cut-and-paste mechanism. Therefore, only class II elements, transposons, cause mutable traits by precise excision from the silent alleles where they are inserted. In rice (Oryza sativa L.), retrotransposons were detected as active transposable elements (Hirochika et al. 1996), and their application to rice genomics and genetics has advanced considerably (Hirochika 2001). An active transposon was not detected for a long time in the rice genome. However, now we know of two such transposons. A number of analyses of maize mutable phenotypes caused by transposons have led to the grouping of transposons into several families (Peterson 1988), such

The early flowering trait of an emmer wheat accession (Triticum turgidum L. ssp. dicoccum) is associated with the cis-element of the Vrn-A3 locus

Key messageWe identified a novel allele of the Vrn-A3 gene that is associated with an early flowering trait in wheat. This trait is caused by a cis-element GATA box in Vrn-A3.AbstractTo identify novel flowering genes in wheat, we investigated days from germination to heading (DGH) in tetraploid wheat accessions. We found that the tetraploid variety Triticum turgidum L. ssp. dicoccum (TN26) harbors unknown genes that surpass the earliness effect of the early flowering allele Ppd-A1a harbored by TN28 (T. turgidum L. ssp. turgidum conv. pyramidale). Using recombinant inbred lines resulting from a cross between TN26 and TN28, we performed a quantitative trait locus (QTL) analysis for DGH. We identified a QTL for earliness in TN26 on chromosome 7AS, the chromosome on which Vrn-A3 is located. By sequence analysis for the Vrn-A3 locus in both TN26 and TN28, we identified a 7-bp insertion that included a cis-element GATA box sequence at the promoter region of the Vrn-A3 locus of TN26. Based on an expression analysis using sister lines for Vrn-A3, we suggest that the early flowering trait of TN26 was caused by the GATA box in Vrn-A3. In addition, we identified tetraploid wheat as a useful genetic resource for wheat breeding.