Yasuo Kowyama

Molecular and genetic characterization of the S locus in Hordeum bulbosum L., a wild self-incompatible species related to cultivated barley.

Gametophytic self-incompatibility (GSI) in the grasses is controlled by a distinct two-locus genetic system governed by the multiallelic loci S and Z. We have employed diploid Hordeum bulbosum as a model species for identifying the self-incompatibility (SI) genes and for elucidating the molecular mechanisms of the two-locus SI system in the grasses. In this study, we attempted to identify S haplotype-specific cDNAs expressed in pistils and anthers at the flowering stage in H. bulbosum, using the AFLP-based mRNA fingerprinting (AMF, also called cDNA-AFLP) technique. We used the AMF-derived DNA clones as markers for fine mapping of the S locus, and found that the locus resided in a chromosomal region displaying remarkable suppression of recombination, encompassing a large physical region. Furthermore, we identified three AMF-derived markers displaying complete linkage to the S locus, although they showed no significant homology with genes of known functions. Two of these markers showed expression patterns that were specific to the reproductive organs (pistil or anther), suggesting that they could be potential candidates for the S gene.

SLG/SRK-like genes are expressed in the reproductive tissues of Ipomoea trifida

Self-incompatibility of Ipomoea trifida is under the control of a single multi-allelic locus, called the S-locus, and is genetically the same as the sporophytic system found in some species of Brassica. We investigated whether the S-locus-specific glycoprotein (SLG) and S receptor kinase (SRK) genes associated with the self-incompatibility of Brassica are expressed in the reproductive tissues of I. trifida. We amplified mRNAs from reproductive tissues of I. trifida by reverse transcriptase PCR using primers corresponding to conserved regions of the Brassica SLGs. Four kinds of PCR fragments were amplified in this experiment. These fragments were designated IPG1 to IPG4 based on dot-blot cross-hybridization. Nucleotide sequencing of the clones revealed 40–46% similarity to the Brassica SLGs and SRKs at the protein level. Northern analysis using IPG1 as a probe revealed a major transcript of 2.8 kb that would correspond in size to a fully spliced SRK transcript of Brassica. The transcript for IPG1 was detected in both mature stigma and anther tissue and was developmentally regulated. The experimental results reported here indicate that at least four kinds of SLG/SRK-like genes are expressed in the reproductive tissues of Ipomoea.

Visualization of the S-locus region in Ipomoea trifida: toward positional cloning of self-incompatibility genes

Self-incompatibility (SI) in Ipomoea trifida is regulated by a single S locus with multiple alleles. Identification of SI genes in the S-locus region by positional cloning is one of the most important goals for understanding sexual reproduction in this species. Despite our intensive efforts to construct bacterial artificial chromosome (BAC) contigs covering the S-locus region, a gap was observed in the core region of the potential S locus. In order to confirm the physical linkage of two non-overlapping BAC contigs in the S-locus region and to determine the size of the gap between them, fluorescence in-situ hybridization (FISH) was performed on mitotic chromosomes and extended DNA fibres using previously isolated S-linked BAC clones as probes. The information obtained from this work would be useful for molecular cloning of the SI genes by a chromosome walking approach. In addition, we showed that strong suppression of recombination in the S locus was not related to the centromere because the S locus was mapped to one end of a chromosome.

Molecular Genetics of Sporophytic Self-Incompatibility in Ipomoea, aMember of the Convolvulaceae

Diploid Ipomoea trifida in the Convolvulaceae is a close relative of the cultivated hexaploid species, the sweet potato, and has sporophytic self-incompatibility controlled by a single multi-allelic S-locus. Genetic analyses of I. trifida plants collected from native populations in Central America have identified a number of different S-haplotypes, which show a linear dominance hierarchy with some codominance relationships. A linkage map of DNA markers showed that the S-locus is delimited to a 0.23 cM region and is located in the S-haplotype-specific divergent region (SDR) that has a physical size of 35–95 kb. Of the six genes located within the SDR, three stigma-specific novel genes, SE1, SE2 and SEA, and an antherspecific gene, AB2, are candidates for encoding pistil and pollen determinants of self-incompatibility, respectively, suggesting that a unique recognition mechanism is involved in the self-incompatibility system of Ipomoea.

A self-compatible mutant S allele conferring a dominant negative effect on the functional S allele in Ipomoea trifida

Abstract A spontaneously occurring self-compatible mutant has been identified in Ipomoea trifida, a species possessing sporophytic self-incompatibility controlled by a single multiallelic S locus. Analysis of the segregation of compatibility/incompatibility phenotypes in selfed and crossed progenies of the self-compatible mutant plant indicated that the self-compatibility trait was caused by a mutation at the S locus; the mutated S allele was therefore designated Sc. RFLP analysis of progeny plants segregating for the Sc allele using the SSP gene (a gene linked closely to the S locus of I. trifida) as a probe confirmed that the mutation was present at the S locus. Self-incompatibility responses were examined in F1 progenies obtained from crosses between the self-compatible mutant and self-incompatible plants homozygous for one of three S alleles, S1, S3 and S22, where the dominance relationship is S22>S1>S3. All F1 progeny plants from crosses with S22 and S1 homozygotes were self-incompatible and exhibited the respective phenotypes of each self-incompatible parent (either S22 or S1) in both stigma and pollen. However, of the F1 progeny plants from the cross with the S3 homozygote, those carrying the genotype ScS3 were all self-compatible and cross-compatible as both female and male parents with the S3 homozygote. These results indicate that the dominance relationship between the four S alleles is: S22>S1>Sc>S3 and so reveal the unexpected finding that the mutated Sc allele is dominant over a functional S3 allele. A possible explanation for this observation is that the gene product encoded by the Sc allele confers a dominant negative effect on the S3 gene product.

Sequences of Ipomoea trifida cDNAs related to the Brassica S-locus genes

Self-incompatibility (SI) in the diploid Ipomoea trifida (Convolvulaceae) is sporophytically controlled by a single multiallelic S locus (Kowyama et al. 1980, 1994). In our previous study performed by reverse transcriptase PCR amplification of mRNAs from stigmas and anthers of I. trifida (Kowyama et al. 1995), we detected four kinds of PCR fragment clones (IPG1 to IPG4) with sequence similarity to the S-locus glycoprotein (SLG) and S-receptor kinase (SRK) genes associated with the sporophytic SI of Brassica (Nasrallah and Nasrallah 1993). In subsequent screening of a stigma cDNA library using one of the clones (IPG1) as a probe, we isolated a cDNA clone encoding a putative receptor protein kinase (IRK1) that shares ca. 50% amino acid sequence identity with Brassica SRKs (Kowyama et al. 1996). Restriction fragment length polymorphism (RFLP) analysis, however, showed that neither IRK1 nor the other three genes coding for IPG2 to IPG4 were linked to the S locus of I. trifida (Kowyama et al. 1996). We report here the cloning and sequencing of cDNAs for the latter three genes. A cDNA library (Lambda Zap II) was constructed with poly(A) + RNAs of mature stigmas from a single

Intervarietal variations in radiosensitivity of dry rice seeds irradiated with sparsely and densely ionizing radiation

22 homozygous plant. From screening of the stigma cDNA library using IPG2, IPG3 and IPG4 as probes, one, three and one positively hybridizing clones, respectively, were isolated, cDNA inserts of these clones were subcloned into Bluescript plasmids and sequenced. All of the cDNA clones obtained showed sequences with structural similarity to Brassica SLGs but lacked sequences corresponding to either the transmembrane or ki-

A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription

Abstract Intervarietal differences in radiosensitivity of rice seeds with 12.3% moisture content were examined in terms of chromosomal aberration frequency, mitotic index and root length at seed germination stage, survival rate at seedling stage, M 1 plant fertility at maturity, and chlorophyll mutation rate in M 2 generation after exposures with X- or γ-rays and thermal neutrons. A high correlation of the γ-ray sensitivity with neutron sensitivity was noticed in six rice varieties, suggesting that varietal differences in radiosensitivity of rice seeds can be ascribed to differences in a genetically controlled system, rather than to environmental and physiological factors such as water and oxygen contents in seeds. Conspicuous varietal differences were observed in chromosomal aberration frequency, mitotic index and root growth reduction, whereas the M 1 plant fertility and chlorophyll mutation rate exhibited no varietal differences, indicating that the disappearance of varietal differences measured by the latter two criteria could be due to somatic and/or gametic elimination of aberrant cells during developmental stages after seed irradiation. The present study points out that radiation-induced chromosomal aberrations and mitotic inhibition play an important role for determining the intervarietal differences in radiosensitivity of rice seeds. RBE values of thermal neutrons for root growth reduction and seed fertility in M 1 plants were 11.1 and 14.1 on average, respectively. There was no varietal difference in RBE values among the rice varieties.