Takashige Ishii

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Abstract Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species.

OsLG1 regulates a closed panicle trait in domesticated rice

Reduction in seed shattering was an important phenotypic change during cereal domestication. Here we show that a simple morphological change in rice panicle shape, controlled by the SPR3 locus, has a large impact on seed-shedding and pollinating behaviors. In the wild genetic background of rice, we found that plants with a cultivated-like type of closed panicle had significantly reduced seed shedding through seed retention. In addition, the long awns in closed panicles disturbed the free exposure of anthers and stigmas on the flowering spikelets, resulting in a significant reduction of the outcrossing rate. We localized the SPR3 locus to a 9.3-kb genomic region, and our complementation tests suggest that this region regulates the liguleless gene (OsLG1). Sequencing analysis identified reduced nucleotide diversity and a selective sweep at the SPR3 locus in cultivated rice. Our results suggest that a closed panicle was a selected trait during rice domestication.

Comparative molecular mapping in Ceratotropis species using an interspecific cross between azuki bean (Vigna angularis) and rice bean (V. umbellata).

Abstract A genetic linkage map was developed with 86 F2 plants derived from an interspecific cross between azuki bean (Vigna angularis, 2n=2x=22) and rice bean (V. umbellata, 2n=2x=22). In total, 14 linkage groups, each containing more than 4 markers, were constructed with one phenotypic, 114 RFLP and 74 RAPD markers. The total map size was 1702 cM, and the average distance between markers was 9.7 cM. The loci showing significant deviation from the expected ratio clustered in several linkage groups. Most of the skewed loci were due to the predominance of rice bean alleles. The azuki-rice bean linkage map was compared with other available maps of Vigna species in subgenus Ceratotropis. Based on the lineage of the common mapped markers, 7 and 16 conserved linkage blocks were found in the interspecific map of azuki bean ×V. nakashimae and mungbean map, respectively. Although the present map is not fully saturated, it may facilitate gene tagging, QTL mapping and further useful gene transfer for azuki bean breeding.

A genetic linkage map of azuki bean constructed with molecular and morphological markers using an interspecific population (Vigna angularis x V. nakashimae)

A genetic linkage map of azuki bean (Vigna angularis) was constructed with molecular and morphological markers using an F2 population of an interspecific cross between azuki bean and its wild relative, V. nakashimae. In total, 132 markers (108 RAPD, 19 RFLP and five morphological markers) were mapped in 14 linkage groups covering 1250 cM; ten remained unlinked. The clusters of markers showing distorted segregation were found in linkage groups 2, 8 and 12. By comparing the azuki linkage map with those of mungbean and cowpea, using 20 RFLP common markers, some sets of the markers were found to belong to the same linkage groups of the respective maps, indicating that these linkage blocks are conserved among the three Vigna species. This map provides a tool for markerassisted selection and for studies of genome organization in Vigna species.

Genetic diversity and phylogeny of Japanese sake-brewing rice as revealed by AFLP and nuclear and chloroplast SSR markers.

Japanese rice (Oryza sativa L.) cultivars that are strictly used for the brewing of sake (Japanese rice wine) represent a unique and traditional group. These cultivars are characterized by common traits such as large grain size with low protein content and a large, central white-core structure. To understand the genetic diversity and phylogenetic characteristics of sake-brewing rice, we performed amplified fragment length polymorphism and simple sequence repeat analyses, using 95 cultivars of local and modern sake-brewing rice together with 76 cultivars of local and modern cooking rice. Our analysis of both nuclear and chloroplast genome polymorphisms showed that the genetic diversity in sake-brewing rice cultivars was much smaller than the diversity found in cooking rice cultivars. Interestingly, the genetic diversity within the modern sake-brewing cultivars was about twofold higher than the diversity within the local sake-brewing cultivars, which was in contrast to the cooking cultivars. This is most likely due to introgression of the modern cooking cultivars into the modern sake-brewing cultivars through breeding practices. Cluster analysis and chloroplast haplotype analysis suggested that the local sake-brewing cultivars originated monophyletically in the western regions of Japan. Analysis of variance tests showed that several markers were significantly associated with sake-brewing traits, particularly with the large white-core structure.

Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice

We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube, one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions. The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200–300 individuals in a few hours. These features enabled us to perform rapid largescale seedling genotyping required for marker-assisted selection. We have also examined the applicability of this method for other PCR-based markers: RAPDs, nuclear STS, chloroplast STS and chloroplast microsatellites.

Genetic Analysis of Long Chain Synthesis in Rice Amylopectin

The amount of long chains (LC) of amylopectin in high-amylose rice is thought to be one of the important determinants of its quality when cooked. A wide range of differences in LC content have been reported in rice varieties, which can be clearly divided into four classes based on LC and apparent amylose content: namely, amylose and LC-free, low or medium-amylose and low-LC, high-amylose and medium-LC, high-amylose and high-LC. However, genetic factors controlling LC content have not been fully understood. Here, we performed quantitative trait loci (QTL) analysis of LC content using 157 recombinant inbred lines (RILs) derived from a cross of a low-LC cultivar, Hyogokitanishiki, and a high-LC line, Hokuriku 142. By analyzing randomly selected 15 RILs, it was shown that high LC content (≥11%) was associated with high setback viscosity (≥200 RVU), and that low LC (≤ 3%) was associated with low setback viscosity (≤ 130 RVU), as measured by a Rapid Visco Analyzer. With setback viscosity as an indicator for LC content, QTL analysis was conducted using 60 DNA markers including a CAPS marker that distinguished Wxa and Wxb alleles coding for granule-bound starch synthase I (GBSSI or Wx protein), the enzyme working for amylose biosynthesis. Only one QTL with a peak log of likelihood score at the wx locus was detected, and no line showing setback viscosity corresponding to the medium-LC class appeared. The fact that wx mutants of Hokuriku 142 lacked LC in their rice starch supports the view that the functional Wx allele is indispensable for LC synthesis in addition to amylose synthesis in rice endosperm. We suggest three possible reasons why no line with medium-LC content was observed. First, the locus (loci) responsible for generation of medium-LC may be located very close to the wx locus and not able to be dissected by the population and DNA markers we used. Second, there may be special QTLs for medium-LC cultivars that do not exist in low- or high-LC cultivars. Third, medium-LC cultivars may have an as-yet unidentified Wx allele with lower capability in LC synthesis compared to the Wx allele in high-LC cultivars.

Nucleotide polymorphism in the Adh1 locus region of the wild rice Oryza rufipogon.

Nucleotide variation in the alcohol dehydrogenase (Adh1) locus region of the wild rice Oryza rufipogon and its related species was analysed to clarify the maintenance mechanism of DNA variation in Oryza species. The estimated nucleotide diversity in the Adh1 locus region of O. rufipogon was 0.002, which was one of the lowest values detected in nuclear loci of plant species investigated so far. Tests of neutrality detected significantly negative deviation from the neutral mutation model for the coding region, especially for replacement sites. When each of the ADH1 domains was considered, significance was detected only for the catalytic domain 1. These results suggest purifying selection in the Adh1 coding region. In the phylogenetic tree of Oryza species based on Adh1 variation, cultivated rice O. sativa subspp. japonica and indica were included in the cluster of O. rufipogon. The genetic distance of the Adh1 region between O. rufipogon and O. sativa was as low as the nucleotide diversity of O. rufipogon. These results imply that O. rufipogon and O. sativa cannot be classified based on the nucleotide variation of Adh1. No replacement divergence between O. rufipogon and the other three A-genome species (O. glumaepatula, O. barthii and O. meridionalis) were detected, indicating that ADH1 is conserved in the A-genome species. On the other hand, between O. rufipogon and the E-genome species O. australiensis, replacement changes were detected only in the catalytic domain 1. The difference in replacement substitutions between the A- and E-genome species may be related to adaptive changes in the ADH1 domains, reflecting environmental differences where the species encounter anaerobic stress.

Germplasm Release: Saikai 35, a Male and Female Fertile Breeding Line Carrying Solanum Phureja-Derived Cytoplasm and Potato Cyst Nematode Resistance (H1) and Potato Virus Y Resistance (Rychc) Genes

Saikai 35 was bred from a cross between TD0101 as the female, which was created by chromosome-doubling of a good-tasting and bacterial wilt resistant diploid variety, Inca-no-mezame, and Sakurafubuki as the male, the latter of which has H1 and Rychc genes showing resistance to potato cyst nematode (PCN) and Potato virus Y (PVY), respectively. All favorable traits were combined into Saikai 35, although marketable yield in the spring cropping was 20.4–21.0% lower than those of major double-cropping varieties. Saikai 35 is particularly useful for having Solanum phureja-derived cytoplasm (S/ε), which resulted in high male and female fertility. In addition, sets of very tightly linked DNA markers sandwiching H1 and Rychc are available. Therefore, Saikai 35 is being released as a breeding line, which can confer efficiently PCN and PVY resistance genes.ResumenSaikai 35 es el resultado de la cruza entre TD0101 como hembra, que se creó por duplicidad de cromosomas de una variedad diploide de buen sabor y resistente a la marchitez bacteriana, Inca-no-mezame, y Sakurafubuki como macho, que tiene genes de H1 y Rychc que muestran resistencia al nematodo de quiste de la papa (PCN) y al virus Y de la papa (PVY), respectivamente. Se combinaron todos los caracteres favorables en Saikai 35, aunque el rendimiento comercial en el cultivo de primavera fue 20.4–21.0% más bajo que las principales variedades de doble cultivo. Saikai es particularmente útil por tener citoplasma derivado de Solanum phureja (S/ε), que da como resultado alta fertilidad masculina y femenina. Además, están disponibles juegos de marcadores de DNA fuertemente ligados en sándwich con H1 y Rychc. De aquí que Saikai 35 se está liberando como una línea de mejoramiento, que puede conferir eficientemente genes de resistencia a PCN y PVY.

Enhancement of Rice Leaf Photosynthesis by Crossing between Cultivated Rice, Oryza sativa and Wild Rice Species, Oryza rufipogon

Abstract To study whether wild rice species have genes that may increase potential photosynthetic capacities of rice cultivars, we generated BC2 populations by reciprocally backcrossing Oryza rufipogon (W630) with O. sativa cv. Nipponbare and IR36; N-BC2 populations and IR-BC2 populations, respectively. We measured the oxygen evolution rates (OER) of single leaves under saturating light and CO2 as the maximum photosynthetic rates and the contents of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase. Several lines in each BC2 population had significantly higher OERs than parental cultivars, and 14~25% of plants inBC2 populations had higher OERs than the highest values in parental cultivars. The highest OERs in BC2 populations were about 60% higher than average OERs in parental cultivars. The BC2 populations contained 30~40% more Rubisco than parental cultivars. The Rubisco activase contents in N-BC2 populations were 15~30% lower than that in Nipponbare. Cytoplasms derived from O. rufipogon and O. sativa had different effects on the contents of Rubisco and Rubisco activase particularly in N-BC2 populations. In several lines of each BC2 population the OERs had positive correlations with the contents of Rubisco and/or Rubisco activase. These results suggest that O. rufipogon can be used as a source of germplasm to enhance the photosynthetic capacity of O. sativa.