Ryuji Ishikawa

Chloroplast genome sequence confirms distinctness of Australian and Asian wild rice

Cultivated rice (Oryza sativa) is an AA genome Oryza species that was most likely domesticated from wild populations of O. rufipogon in Asia. O. rufipogon and O. meridionalis are the only AA genome species found within Australia and occur as widespread populations across northern Australia. The chloroplast genome sequence of O. rufipogon from Asia and Australia and O. meridionalis and O. australiensis (an Australian member of the genus very distant from O. sativa) was obtained by massively parallel sequencing and compared with the chloroplast genome sequence of domesticated O. sativa. Oryza australiensis differed in more than 850 sites single nucleotide polymorphism or indel from each of the other samples. The other wild rice species had only around 100 differences relative to cultivated rice. The chloroplast genomes of Australian O. rufipogon and O. meridionalis were closely related with only 32 differences. The Asian O. rufipogon chloroplast genome (with only 68 differences) was closer to O. sativa than the Australian taxa (both with more than 100 differences). The chloroplast sequences emphasize the genetic distinctness of the Australian populations and their potential as a source of novel rice germplasm. The Australian O. rufipogon may be a perennial form of O. meridionalis.

DNA-RAPDs detect genetic variation and paternity in Malus

SummaryDNA amplification fingerprinting using arbitrary primer(s) was applied to the identification of Malus species. Highly variable DNA fragment patterns were clearly detected by polyacrylamide gel electrophoresis of the amplified extension products, although three sports of Delicious exhibited the same fingerprint as the original cultivar. The fingerprinting of two triploid apple cultivars suggested that the parent contributing the 2n gamete was the female. We applied this fingerprinting to paternity analysis of an apple cultivar of which the pollen parent was unknown. By using 5 arbitrary primers and RFLP analysis of the amplified products, one cultivar was singled out for paternity among six putative candidates.

Origin of Weedy Rice Grown in Bhutan and the Force of Genetic Diversity

In Bhutan, weedy rice (Oryza sativa L.) was grown together with cultivated rice on terraced paddy fields lower than 2620 m above sea level. Seeds of cultivars and weedy strains were collected at 22 collection sites located from 1000 to 2620 m above sea level. Cultivars with round seeds were frequently found in fields higher than 2250 m, and those with slender seeds in fields lower than 1630 m. All cultivars and weedy strains were divided into indica or japonica types by isozyme (multi-locus) and morpho-physiological (multicharacter) analyses. Japonica cultivars predominated in highland; Indica cultivars predominated in lowland. Plastid type was confirmed by the length polymorphism for the ORF100 region. The japonica cultivars carried non-deletion type ORF100. The indica cultivars carried deletion type ORF100. In contrast, weedy strains showed discrepancy in the combination of the nuclear and cytoplasm types. An intermediate type was found in weedy strains for isozyme genotypes. A recombinant type, which has indica genotypes for isozyme analysis with japonica cytoplasm, and vice versa, was frequently found in weedy strains. These findings suggested that weedy strains would be generated by natural hybridization between indica and japonica. Further, they might fail to recombine their prior genotypes sets for isozyme and cytoplasm. Morphophysiological characters did not show such a tendency. Morpho-physiological analysis of highland plants, in particular, showed indica cultivars and weedy strains with japonica cytoplasm. Hypervariable nuclear microsatellite analysis was then used to compare cultivars and weedy strains. Identical alleles were shared between indica and japonica, and also between cultivars and weedy strains. This suggested that there was gene-flow resulting from natural hybridization.

Australian Oryza: utility and conservation

Australian Oryza are an understudied and underexploited genetic resource for rice improvement. Four species are indigenous: Oryza rufipogon, Oryza meridionalis, Oryza australiensis are widespread across northern Australia, whereas Oryza officinalis is known from two localities only. Molecular analysis of these wild populations is required to better define the distinctness of the taxa and the extent of any gene flow between them and rice. Limited collections of these wild populations are held in seed and DNA banks. These species have potential for domestication in some cases but also have many traits of potential value in the improvement of domesticated rice. Stress tolerance (biotic and abiotic) and grain quality characteristics in these populations may be useful.

Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth

SummaryAlbino rice plants derived from pollen contain plastid genomes that have suffered large-scale deletions. From the roots of albino plants, we obtained several calli containing homogeneous plastid DNA differing in the size and position of the deletion. Southern blotting and pulsed field gel electrophoresis experiments revealed that the DNAs were linear molecules having a hairpin structure at both termini, existing as monomers (19 kb) or dimers, trimers and tetramers linked to form head-to-head and tail-to-tail multimers. This characteristic form is similar to that of the vaccinia virus, in which the replication origin is thought to lie at or near the hairpin termini. Furthermore, polymerase chain reaction experiments revealed complete loss of the ribosomal RNA genes of the plastid DNA. The results suggest that plant cells can grow without translation occurring in plastids. All of the deleted plastid DNAs commonly retained the region containing the tRNAGlu gene (trnE), which is essential for biosynthesis of porphyrin. As porphyrin is the precursor of heme for mitochondria and other organelles, it is considered thattrnE on the remnant plastid genome may be transcribed by an RNA polymerase encoded on nuclear DNA.

Analysis of the duplicated CHS1 gene related to the suppression of the seed coat pigmentation in yellow soybeans

Abstract.Seed coat color in soybean is controlled by the classically defined I (Inhibitor) locus. The seeds of most commercial soybean varieties are yellow due to the presence of a dominant allele of the I locus (I: yellow seed coat, or ii : pigmented hilum and yellow seed coat), which inhibits seed coat pigmentation. Analysis of spontaneous mutations from I (yellow seed coat) to i (pigmented seed coat) has shown that these mutations are correlated with the deletion of a duplicated chalcone synthase gene-1 (CHS1) region. In the current study, we isolated the duplicated CHS1 region from a soybean cultivar with a I/I genotype (cv Miyagi shirome) and determined its structure. The results showed that the duplicated CHS1 contained intact regulatory and coding regions. We designated the duplicated CHS1 as ICHS1. In the hypocotyls of Miyagi shirome, the cDNA derived from ICHS1 mRNA was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, whereas in the immature seed coats it was suggested that the amount of transcripts from ICHS1 and/or another type of CHS1 (CHS1.1) was very low. Interestingly, in the Miyagi shirome genome with a I/I genotype, ICHS1 was closely linked to the truncated CHS3, and sequence comparison showed that this cluster probably arose from the CHS1–CHS3 cluster by a 1.8-kb deletion event.

Isolation of a subfamily of genes for R2R3-MYB transcription factors showing up-regulated expression under nitrogen nutrient-limited conditions

Plant R2R3-MYB transcription factors are encoded by more than 100 copies of genes. In this study, we attempted to isolate some members of the R2R3-MYB superfamily involved in regulation of nitrogen fixation in legumes. A library of 300 recombinant plasmid clones containing the R2R3-MYB fragments of the superfamily was screened by differential hybridization to isolate R2R3-MYB genes whose expression was up-regulated under nitrogen nutrient-limited conditions. Two groups of clones were identified, each of which seemed to represent a gene responsive to nitrogen starvation. The entire coding regions for the genes were further isolated by PCR and were designated LjMYB101 and LjMYB102. By screening a genomic library of Lotus japonicus with a probe derived from LjMYB101, the third gene, LjMYB103, was isolated. In addition, a candidate for the soybean orthologue of LjMYB101 was isolated and designated GmMYB101. Sequence alignment of the genes with members of the plant R2R3-MYB superfamily showed that they all belonged to the subgroup 10 of the superfamily. The expression analysis of the genes showed that the organ-specific and nitrate-regulated expression profile of MYB101 was very similar to that of CHS in Lotus as well as in soybean, suggesting a possible role for MYB101 in regulation of flavonoid biosynthesis in response to nitrate starvation. On the other hand, an interesting relationship, in structure and function, was found between LjMYB101 and LjGln1, suggesting an alternative role for MYB101 in regulation of nitrogen metabolism.

Different maternal origins of Japanese lowland and upland rice populations

Abstract.Plastid subtype ID (PS-ID) sequences were determined from sequence data based on CA repeats between genes rpl16 and rpl14 in Japanese lowland and upland cultivars. The PS-ID sequences of Japanese rice cultivars showed that there are different maternal origins between lowland and upland cultivars. One subtype, 6C7A, of PS-ID sequences was predominant in all but one Japanese lowland cultivar and carried a combination of the indica-specific subtype 8C8A and japonica-specific nuclear markers for the isozyme genotype. It is probably a nuclear-cytoplasmic recombinant resulting from natural out-crossing and succeeding self-pollination. The origin of the plastid was re-confirmed by the existence of an indica-specific deletion in the plastid genome. In contrast, the Japanese upland cultivars showed two subtypes, 7C6A and 6C7A, of PS-ID sequences. An upland-specific isozyme allele as a nuclear marker was equally predominant in cultivars carrying each subtype. The existence of these particular upland-specific nuclear and cytoplasmic genotypes suggests that the origin of Japanese upland cultivars is different from that of Japanese lowland cultivars. Cultivars carrying the upland-specific nuclear genotype are common in Southeast Asia, but the combination of the upland-specific nuclear and cytoplasmic genotypes which is the same as the Japanese upland predominant type was found in cultivars only in Taiwan and Indonesia. Japanese upland cultivars are closely related to those cultivars.

Molecular relationships between Australian annual wild rice, Oryza meridionalis, and two related perennial forms

BackgroundThe perennial, Oryza rufipogon distributed from Asia to Australia and the annual O. meridionalis indigenous to Australia are AA genome species in the Oryza. However, recent research has demonstrated that the Australian AA genome perennial populations have maternal genomes more closely related to those of O. meridionalis than to those found in Asian populations of O. rufipogon suggesting that the Australian perennials may represent a new distinct gene pool for rice.ResultsAnalysis of an Oryza core collection covering AA genome species from Asia to Oceania revealed that some Oceania perennials had organellar genomes closely related to that of O meridionalis (meridionalis-type). O. rufipogon accessions from New Guinea carried either the meridionalis-type or rufirpogon-type (like O. rufipogon) organellar genomes. Australian perennials carried only the meridionalis-type organellar genomes when accompanied by the rufipogon-type nuclear genome. New accessions were collected to better characterize the Australian perennials, and their life histories (annual or perennial) were confirmed by field observations. All of the material collected carried only meridionalis-type organellar genomes. However, there were two distinct perennial groups. One of them carried an rufipogon-type nuclear genome similar to the Australian O. rufipogon in the core collection and the other carried an meridionalis-type nuclear genome not represented in the existing collection. Morphologically the rufipogon-type shared similarity with Asian O. rufipogon. The meridionalis-type showed some similarities to O. meridionalis such as the short anthers usually characteristic of annual populations. However, the meridionalis-type perennial was readily distinguished from O. meridionalis by the presence of a larger lemma and higher number of spikelets.ConclusionAnalysis of current accessions clearly indicated that there are two distinct types of Australian perennials. Both of them differed genetically from Asian O. rufipogon. One lineage is closely related to O. meridionalis and another to Asian O. rufipogon. The first was presumed to have evolved by divergence from O. meridionalis becoming differentiated as a perennial species in Australia indicating that it represents a new gene pool. The second, apparently derived from Asian O. rufipogon, possibly arrived in Australia later.

Characterization of a DMC1 homologue, RiLIM15, in meiotic panicles, mitotic cultured cells and mature leaves of rice (Oryzasativa L.)

Abstract DMC1 is one of the most important genes involved in meiotic homologous recombination in Saccharomyces cerevisiae. Homologues of DMC1 have been isolated recently from some plant species, and in this study, we characterized the structure and expression of a DMC1 homologue, RiLIM15, in a Japonica rice, strain A58. RiLIM15 was found to be a gene family consisting of two genes, RiLIM15A and RiLIM15B, in the rice genome. The DNA sequence of RiLIM15A was highly homologous with that of RiLIM15B in the exon regions, although it was less homologous with that of RiLIM15B in the intron regions. Analysis for the expression of RiLIM15 by a combination of Southern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) showed that RiLIM15 was expressed not only in meiotic young panicles, but also in mitotic cultured cells, although not in the mature leaves. Analysis of the sequences of these RiLIM15 cDNAs amplified by RT-PCR showed that the sequences of exon 5 were deleted from the cDNAs derived from the meiotic young panicles. Also, exons 5, 10 and 11, as well as 29 bp of exon 8, were deleted from some types of cDNA from the mitotic cultured cells. These results suggest that these deletions may be caused by alternative splicing.