Toshiro Kinoshita

Genomic organization and sequence analysis of the cytochrome oxidase subunit II gene from normal and male-sterile mitochondria in sugar beet.

SummaryWe have cloned and sequenced the cytochrome oxidase subunit II (coxII) gene from both normal and cytoplasmic male-sterile (CMS) sugar beet. The normal coxII (designated NcoxII) locus was found to be located 1491 bp upstream from the gene for cytochrome oxidase subunit I (coxI) on the same DNA strand and to have a 1463 bp intron which split the coding sequence into two exons (382 and 398 bp). The COXII protein contains 260 amino acid residues. We have also found two copies of the coxII gene (ScoxII-1 and ScoxII-2) to be present in the CMS genome. Our results suggest that the NcoxII gene diverges completely from the ScoxII-1 and ScoxII-2 genes 50 bp 5′ to the ATG start codon. In addition, the ScoxII-1 and ScoxII-2 sequences could be readily discriminated from each other by the 3′ end and the immediately adjacent flanking sequences of the gene: the 3′ divergence results in a 101 codon extension of the ScoxII-2 ORF. Northern blot analysis demonstrates that the coxII gene exhibits altered transcript patterns in CMS compared with normal sugar beet. Different genomic arrangements of the coxII gene are considered to be the result of extensive intra-and inter-molecular recombination events involving the repeated DNA elements in the mitochondrial genome.

Organelle genome diversity in sugar beet with normal and different sources of male sterile cytoplasms.

SummaryMitochondrial (mt) and chloroplast (ct) DNAs from sugar beet lines carrying normal and introduced sources of male sterile cytoplasms have been characterized and compared on the basis of restriction enzyme analysis. Normal cytoplasm was shown to contain mt and ctDNAs which differed from those of the male sterile cytoplasms examined in the present investigation. On the other hand, four groups of male sterile cytoplasms could be differentiated by their own characteristic mtDNA digest patterns, while two were separated by ctDNA comparisons. In addition, a greater degree of variability of the mitochondrial genome is suggested. Our results also imply strict maternal inheritance of mt and ctDNAs. Thus, the organelle DNA assay provides a positive and alternative means of identifying various male sterile cytoplasms.

Development of pre-isogenic lines for rice blast-resistance by marker-aided selection from a recombinant inbred population.

To increase the available set of near-isogenic lines (NILs) for blast-resistance in rice, we have developed a general method for establishing NILs from populations of fixed recombinants that have been used for gene mapping. We demonstrated the application of this method by the selection of lines carrying genes from the rice cultivar Moroberekan. Moroberekan is a West African japonica cultivar that is considered to have durable resistance to rice blast. Multiple genes from Moroberekan conferring complete and partial resistance to blast have previously been mapped using a recombinant inbred (RI) population derived from a cross between Moroberekan and the highly and broadly susceptible indica cultivar CO39. To analyze individual blast-resistance genes, it is desirable to transfer them individually into a susceptible genetic background. This RI population, and the associated data sets on blast reaction and restriction fragment length polymorphism (RFLP) genotypes, were used for selection of lines likely to carry individual blast-resistance genes and a minimum number of chromosomal segments from Moroberekan. Because skewed segregation in the RI population favored CO39 (indica) alleles, resistant lines carrying 8.7–17.5% of Moroberekan alleles (the proportion expected after two or three backcrosses) could be selected. We chose three RI lines carrying different complete resistance genes to blast and two RI lines carrying partial resistance genes to blast as potential parents for the development of NILs. These lines were subjected to genetic analysis, which allowed clarification of some issues that could not be resolved during the initial gene-mapping study.

Molecular heterogeneity in mitochondrial and chloroplast DNAs from normal and male sterile cytoplasms in sugar beets

SummaryMitochondrial (mt) and chloroplast (ct) DNAs were prepared from normal (N) and male sterile (S) cytoplasmic lines of sugar beet. The DNAs were cleaved with BamHI, EcoRI, HindIII and SalI enzymes, and the resultant fragments were fractionated by agarose gel electrophoresis. The results showed that N and S cytoplasms contained distinct mtDNA. Although most of the DNA fragments were common to these two cytoplasms, each cytoplasm was readily characterized by bands specific to that cytoplasm. In addition, these distinctive cleavage patterns were invariant in different nuclear backgrounds. In contrast to the marked variation in mtDNA, restriction fragment analyses of ctDNA demonstrated little difference between both cytoplasms. Only HindIII digestion showed one band missing in the S genome. The data presented here provides circumstantial evidence for mitochondrial involvement in the inheritance of cytoplasmic male sterility in sugar beet.

The sugar beet mitochondrial gene for the ATPase alpha-subunit : sequence, transcription and rearrangements in cytoplasmic male-sterile plants

We have characterized the mitochondrial atpA (the alpha subunit of F1-ATPase) gene from male-fertile cytoplasm (cv TK81-0) of sugar beet. The gene is 1518-bp long and encodes a polypeptide of 506 amino acids. The atpA mRNA sequence is modified by three C-to-U RNA editing events, all of which alter the encoded protein sequences. In order to examine the genome organization of the atpA locus in cytoplasmic male-sterile (CMS) sugar beet, atpA-containing clones were isolated from Owen CMS (TK81-MS) and a different source of CMS [I-12CMS(2)] cytoplasm respectively. The sequences of the atpA coding region from TK81-MS and I-12CMS(2) are identical to each other and to the corresponding TK81-0 sequence. However, the TK81-0 and TK81-MS loci diverge completely 47 bp upstream of the initiation codon, resulting in different 5′ transcript termini for the two genes. On the other hand, the point of divergence between the TK81-0 and I-12CMS(2) atpA genes was found to occur after 393 bp 3′ to the TAA stop codon. Our results also show the 3′-flanking sequences of I-12CMS(2) atpA to be present elsewhere in the mitochondrial genomes of TK81-0, TK81-MS and I-12CMS(2), suggesting the possible involvement of these repeated DNA elements in the sequence rearrangements.

Genotypic effects on the callus formation from different explants of rice, Oryza sativa L.

A total of 108 rice varieties were examined for their tissue culture responses. Callus tissues were initiated from the seed, radicle, coleoptile and anther explants. Our results indicated that genotypes differed in the ability to develop vigorously growing callus. The callus growth responses in seed, radicle and coleoptile cultures were intercorrelated, but were not correlated with that in anther culture.

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation

SummaryThe interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.

Identification of the rice D-genome chromosomes by genomic in situ hybridisation

Abstract The 24 rice D-genome chromosomes were identified among the 48 chromosomes of O. latifolia, which comprise the C- and D-genomes, using genomic in situ hybridisation (GISH). The B-genome chromosomes were also discriminated from the C-genome chromosomes in O. minuta (BBCC) by GISH. A comparison of the differences in the fluorescence intensity between the C and D genomes within O. latifolia (CCDD), and between the B and C genomes within O. minuta, indicated that the overall nucleotide-sequence homology between the B and C genomes is less than that between the C and D genomes. The origin of the D genome and the phylogenetic relationship of the D genome among the rice genomes are discussed, based on the results obtained.

In vitro techniques for genomic alteration in rice plants

Because of the explosive increase in world population, a sufficient food supply must be achieved by varietal improvement in the major cereal crops including rice. It is expected that new in vitro techniques incombination with conventional breeding methods may effectively raise the yield potential. On the other hand, there are many environmental problems to be solved world-wide such as, global warming, environmental pollution, ecological destruction, reduction in water supplies and so on. Therefore, it is necessary to rapidly develop new varieties for the future combining of higher yield potential with excellent grain quality, and resistance to both biotic and abiotic stresses for the promotion of sustainable agriculture. Although many efforts have been made to introduce useful traits from wild species to cultivated rice via hybridization, it is still difficult to overcome breeding barriers such as cross incompatibility and hybrid sterility and inviability in practical breeding. Now in vitro techniques are going to make it possible to use genetic manipulation and cell culture and fusion techniques to speed up the breeding process. For sustainable agriculture, it is important to utilize the useful genes from alien species. For this purpose, asymmetric protoplast fusions have already been used successfully to transfer disease resistance in Brassica napus. In this experiment, a high level of resistance to the rice blast disease was transferred from wild species through asymmetric fusions. It is also noted that manipulation of cytoplasmic genomes is possible through asymmetric fusions as shown in the induction of new cytoplasmic male sterility (CMS).

Ubiquity of the genes for components of a NADH dehydrogenase in higher plant chloroplast genomes

Sequences homologous to the six tobacco chloroplast genes, ndhA-F, which correspond to the genes for components of the human mitochondrial NADH dehydrogenase, have been identified in rice, sugar beet and broad bean chloroplast genomes. The locations of the six ndh sequences are essentially conserved among these chloroplast genomes. The results indicate that the ndh sequences are widespread in chloroplast genomes of higher plants.