Yo-Ichiro Sato

Genetic characterization of weedy rice (Oryza sativa L.) based on morpho-physiology, isozymes and RAPD markers

Abstract Weedy rice (Oryza sativa L.) is an important resource for breeding and for studying the evolution of rice. The present study was carried out to identify the genetic basis of the weedy rices distributed in various countries of the world. One hundred and fifty two strains of weedy rice collected from Bangladesh, Brazil, Bhutan, China, India, Japan, Korea, Nepal, Thailand and the USA were tested for variations in six morpho-physiological characteristics and in 14 isozyme loci. Twenty six weedy strains selected from the above materials were assayed for the Est-10 locus, six RAPD loci of the nuclear genome, and one chloroplast locus. From the results of multivariate analysis based on the morpho-physiological characteristics and the isozymes, weedy rice strains were classified into indica and japonica types, and each type was further divided into forms resembling cultivated and wild rice. Thus, four groups designated as I, II, III and IV were identified. Weedy strains of group I (indica-type similar to cultivars) were distributed mostly in temperate countries, group II (indica-type similar to wild rice) in tropical countries, group III (japonica-type similar to cultivars) in Bhutan and Korea, group IV ( japonica-type similar to wild rice) in China and Korea. In group I, classified as indica, several strains showed japonica-specific RAPD markers, while some others had japonica cytoplasm with indica-specific RAPD markers in a heterozygous state at several loci. One weedy strain belonging to group II showed a wild rice-specific allele at the Est-10 locus. However, in groups III and IV, no variation was ound either for the markers on Est-10 or for the RAPD loci tested. Judging from this study, weedy rice of group I might have originated at least partly from gene flow between indica and japonica, whereas that of group II most probably originated from gene flow between wild and cultivated indica rice. Weedy rice of group III is thought to have originated from old rice cultivars which had reverted to a weedy form, and that of group IV from gene flow between japonica cultivars and wild rice having japonica backgrounds.

Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wxa and Wxb, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wxa allele was predominant in non-waxy indica cultivars, whereas the Wxb allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wxa from Wxb have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wxa or Wxb alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wxa allele (AGGT) but not in the Wxb allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wxa- and Wxb-derived alleles, but that the Wxb-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.

Isolation and Characterization of Endophytic Bacteria from Wild and Traditionally Cultivated Rice Varieties

Abstract Endophytic bacteria were isolated from surface-sterilized stems, seeds, and leaf sheaths of wild and traditionally cultivated rice varieties. Phylogenetic analyses based on 16S rDNA revealed a wide divergence among the isolates. However, the most frequently isolated groups were Methylobacterium sp. in the α-subdivision of Proteobacteria, and Curtobacterium sp. in the high G+ C Gram-positive group. Various phenotypic traits that are expected to be involved in the persistence and functions of the bacteria were analyzed: Most of the isolates from rice excreted pectinase, were motile, and showed an osmotic resistance to 0.6 M sucrose. These traits may be involved in endophytic characteristics in rice. About 50% of the isolates showed a cellulase activity. A few isolates fixed nitrogen, produced indole-3-acetic acid, and formed capsules. These activities were partially correlated with the phylogenetic group.

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.

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.

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.

Gene flow from cultivated rice (Oryza sativa L.) to wild Oryza species (O. rufipogon Griff. and O. nivara Sharma and Shastry) on the Vientiane plain of Laos

Although Asian cultivated rice (Oryza sativa) and its annual (O. nivara) and perennial (O. rufipogon) wild relatives can freely exchange genes under experimental conditions, little is known about gene flow from the cultivated to these two wild species in natural habitats. The area of study was the Vientiane plain of Laos, at the center of the glutinous rice zone. The objectives of this study were (1) to determine the extent of hybridization between rice and its close wild relatives over a large area on the basis of the O. sativa specific traits glutinous endosperm, colorless apiculus and white pericarp; (2) to evaluate the genetic variation in the hybrid plants based on 13 quantitative traits using principal component analysis. Out of 1576 individual plant samples analyzed two hybrid groups were recognized. One hybrid group consisted of O. rufipogon–O. sativa hybrids and represented 10% of wild individual plants analyzed. The other hybrid group was O. nivara–O. sativa hybrids that represented 14% of wild individuals analyzed. These hybrid groups are distinct based on principal component analysis – this is due to both spatial isolation (> 100 m) and reproductive isolation (i.e. differential flowering time). The results indicate that two kinds of gene flow, between (a) O. sativa and O. rufipogon and (b) O. sativa and O. nivara, are occurring throughout the Vientiane plain of Laos.

Genetic Erosion from Modern Varieties into Traditional Upland Rice Cultivars (Oryza sativa L.) in Northern Thailand

The purpose of this study was to assess the extent of genetic erosion of traditional upland germplasm in northern Thailand as a result of gene-flow from distinct strains carrying different genotypes. Even modern variety specific markers have not been developed, there is a comparative population in Laos. Thus, both populations were compared with various characters to evaluate gene-flow from modern variety to landraces. Glutinous and glabrous strains are predominated in Laos. However, such strains were drastically decreased in north–east Thailand. Gene diversity is higher in Thailand, compared to Laos at seven isozyme loci. This was a result of the higher frequencies of Indica strains and heterozygotes in Thailand. Plastid type was also determined by using an INDEL marker. Nearly half of Indica strains carried the Japonica plastid. Heterozygotes also tended to carry Japonica cytoplasm. Such nuclear–cytoplasm substituted strains and heterozygotes were probably generated by natural hybridization. Japonica strains tended to be a maternal donor rather than Indica ones. Or Indica strains would easily release pollens, which grow outside of upland fields.

Genetic resources of primitive upland rice in Laos

Primitive upland cultivars (Oryza sativa L.) were collected in northern Laos. One-hundred-thirty-two cultivars were collected in upland fields at 27 sites. Morphological and physiological traits were recorded. The materials were classified intoindica and japonica types based on isozyme genotypes. We classified 106japonica, 16indica, two intermediate, and eight heterozygous cultivars. Thejaponica cultivars were characterized by glabrous hulls and sticky grains. Only two out of 16indica cultivars were glabrous. The heterozygotes were estimated to be generated by out-crosses betweenjaponica andindica cultivars in upland fields. The intermediate type would be the progeny of such heterozygotes. Higher polymorphism was found at two isozyme loci—Amp1 andEst2—among thejaponica cultivars. Genotypic frequencies differed between populations collected from upland fields along roads and along a branch of the Mekong river. Such differences would be caused by different origins of these two populations. In this report, isozymes were indicated as valuable markers to recognize the cultivars to be of independent stock.

Differential heterosis in a natural population of Asian wild rice (Oryza rufipogon Griff.) due to reproductive strategy and edge effect

Asian common wild rice (Oryza rufipogon) is a polycarpic perennial plant with a mixed inbreeder/outbreeder strategy. Seeds and clones are both reproductive components of the population. However, when collected in the flowering stage, the two differ substantially as to whether they have experienced natural selection directly or not. This study is aimed at evaluating mechanisms for the survival of a population by comparing genetic structure among subpopulations that are classified in terms of: (1) reproductive systems (clones or seeds); and also (2) location (fringe or inside). First, the genotypes determined at seven SSR (Simple Sequence Repeat) loci showed that parameters of observed heterozygosity (HO) and outcrossing rate (t) were clearly higher in clones (HO = 0.609, t = 82.4%) than in seeds (HO = 0.346, t = 35.5%), although the two had approximately the same values for the number of alleles (A) and expected heterozygosity (HE)(A = 4.43 in both clones and seeds, HE = 0.709, 0.692 in clones and seeds, respectively). This result indicates that the individuals showing ’heterosis’, with high numbers of heterozygous loci and outcrossing rates, are more likely to survive in the natural habitat. Secondly, parameters of observed heterozygosity and outcrossing rates were lower for seeds from the fringe area (HO = 0.238, t = 24.6%) compared to seeds from the inside area (HO = 0.443, t = 60.7%), although values were similar in clones of both the fringe and inside areas. This result suggests that the ’edge effect’ might be due mainly to the restriction of wind strength in fringe area of the forested swamp.