RFLP analysis of the progeny from Oryza alta Swallen x Oryza sativa L.

RFLP analyses were carried out in the progeny from a cross of two phylogenetically distant rice species, wild rice Oryza alta Swallen (CCDD, 2n = 48) and cultivated rice O. sativa L. (AA, 2n = 24). The sterile plants gave heterozygous RFLP patterns at most of the loci detected. They looked more like their wild rice parent, with 36 chromosomes in their root-tip cells and pollen mother cells. In two partially fertile plants, however, most of the markers that were used showed RFLP patterns similar to the cultivated parent, O. sativa. By cytological study, it was found that nearly one-third of the chromosomes had been eliminated in the partially fertile plants. Their seeds have short awns, which is a characteristic of their wild parent, O. alta. An introgression occurred in one of the partially fertile plants, which led to the discussion about a nonconventional mechanism in wide hybridization for transference of wild rice chromosome segments to cultivated rice chromosomes.     

普通野生稻（Oryza rufipogon Griff．）的SSR遗传多样性研究

利用SSR标记对分布在我国7个省的17个居群普通野生稻(Oryza rufipogon Griff．)的种质资源进行研究,结果表明：17个居群普通野生稻的遗传多样性有着显著的差异,从UPGMA聚类结果可以看出普通野生稻的遗传多样性与其生态地理分布有显著的相关性,遗传多样性指数高的地区极有可能是栽培稻的起源中心;用筛选出的7对SSR引物对336份：DNA样品进行扩增,得到60条特异条带,在分子水平上进一步证明普通野生稻的起源为两广地区;本研究也表明SSR是进行遗传资源多样性研究的一种切实有效的研究方法。     

Phytochrome action in Oryza sativa L.

Summary The mechanical properties of the cell wall were measured in coleoptiles of totally etiolated rice seedlings. Coleoptiles were either decapitated or briefly exposed to red (R) and/or far-red (FR) light. The elastic and plastic extensibilities of the cell wall changed with age (length) of the coleoptiles. Decapitation and exposure to R induced changes in these properties, and the time-courses were similar. Following decapitation or R irradiation, the plastic extensibility of the cell wall decreased more conspicuously than elastic extensibility. Exogenous application of auxin immediately following decapitation alleviated the effect of removal of the tip. FR irradiation reduced both kinds of extensibilities, but its effect was much less than that of R, and it reversed the R-induced effect to the level of tissue treated with FR only. In repeated R-FR treatments, the decrease of elastic extensibility by R and its reversal by FR could be repeated, but the effect of a second irradiation with R after FR on plastic extensibility was not as apparent as that of the first. Reduction of cell-wall extensibility of etiolated rice coleoptiles caused by R light appeared, at least partly, to be due to a reduced auxin supply in the elongating region from the tip, similar to that caused by decapitation.     

Phytochrome action in Oryza sativa L.

Summary In-vivo phytochrome determinations in totally etiolated rice seedlings with a dual-wavelength spectrophotometer showed that on a fresh weight basis phytochrome concentration was highest in the coleoptile apex (0.175 of mean) ( O.D.) g^-1 (fresh weight). The age of the seedlings had little effect on the pattern of phytochrome distribution in the coleoptiles.The extent of growth inhibition observed 2 days after the irradiations was proportional to the logarithm of P _fr amount in the coleoptiles at the time of initial exposure to either red or blue light. Ultraviolet irradiation, however, did not induce either reversible growth inhibition or optically detectable phytochrome changes in vivo.After the conversion of P _r to P _fr bya brief red irradiation, non-photochemical transformation of phytochrome was observed in intact coleoptile tissues. Most of the optically measurable P _fr disappeared within 6 hours at 27°, when the total ( O.D.) decreased to about one fifth of the original level. The optical data did not agree with the fact that 50% of the initial physiological reversibility was still observed 9 hours later. No significant difference in dark transformation rate was seen between intact and excised coleoptile tissues.Abbreviations P _r red light absorbing form of phytochrome - P _fr far-red light absorbing form of phytochrome - ( O.D.) the change in the optical density difference reading at two wavelengths, following irradiation of the sample with actinic sources of red and far-red light - UV ultraviolet light     

Evaluation of bakanae disease progression caused by Fusarium fujikuroi in Oryza sativa L.

Bakanae disease caused by Fusarium fujikuroi is an important fungal disease in rice. Among the seven strains isolated from symptomatic rice grains in this study, one strain, FfB14, triggered severe root growth inhibition and decay in the crown and root of rice seedlings. The remaining six strains caused typical Bakanae symptoms such as etiolation and abnormal succulent rice growth. To reveal the relationship between mycelial growth in the infected tissues and Bakanae disease progression, we have established a reliable quantification method using real time PCR that employs a primer pair and dual-labeled probe specific to a unigene encoding F. fujikuroi PNG1 (FfPNG1), which is located upstream of the fumonisin biosynthesis gene cluster. Plotting the crossing point (CP) values from the infected tissue DNAs on a standard curve revealed the active fungal growth of FfB14 in the root and crown of rice seedlings, while the growth rate of FfB20 in rice was more than 4 times lower than FfB14. Massive infective mycelial growth of FfB14 was evident in rice stems and crown; however, FfB20 did not exhibit vigorous growth. Our quantitative evaluation system is applicable for the identification of fungal virulence factors other than gibberellin.     

Genetic engineering of Oryza sativa by particle bombardment

Genetic engineering of rice (Oryza sativa L. cv. Pusa basmati 1) using synthetic Cry1Ac gene has been achieved by “particle bombardment”. Scutellar tissues excised after 5 – 6 d from mature seeds cultured on induction medium were bombarded using gold particles coated with a mixture of Cry1Ac and marker genes on medium with osmoticum. Bombarded tissues were subjected to 30 mg dm⁻³ hygromycin selection for two cycles. The selected calli after GUS assay were transferred to shoot regeneration medium. Regenerated shoots were rooted and plantlets (T₀) were grown to full maturity. Polymerase chain reaction (PCR) analysis of T₀ plants using Cry1Ac specific primers revealed the presence of Cry1Ac gene in 65 % plants. Phenotypic assay, β-glucuronidase assay and PCR during T₁ generation revealed the inheritance of the Cry1Ac and marker genes along with the native plant genes.     

Genetic variation in Oryza species detected by MITE-AFLP

MITE-AFLP markers were successfully used to study the genetic variation and species relationship in Oryza species. Analysis of 53 accessions of Oryza species with seven MITE-AFLP primer combinations detected a total of 250 polymorphic fragments. High polymorphism was detected within and between Oryza species. Species relationships were analyzed by the pattern of presence or absence of homologous fragments, because nucleotide sequences of the detected MITE-AFLP fragments revealed that the same fragments in different species shared very high sequence homology. The genetic distances (GDs) between species were higher than those within species and the GDs in O. sativa complex were higher than those in O. officinalis complex. The phylogenetic tree recognized two major groups at 62% genetic similarity; group I consists of all AA genome species of the O. sativa complex, and group II consists of BB-, CC-, EE- and BBCC genome species of the O. officinalis complex. Therefore, this study demonstrated that the MITE-AFLP technique provide a tool for studying the genetic variation and species relationship in Oryza species.     

Induction of somaclonal variation by tissue culture and cytogenetic analysis in Oryza sativa L.

Protocols were developed for plant regeneration from callus induced in mature embryos of rice. Somaclonal variation was scored by genome mutation, chromosome mutation and plasmon mutation in R₀, R₁ and R₂ plant progenies. The frequency of haploids and diploids appeared in the ratio of 20:33. Variation in the chromosome number in callus cells was found to be high and age dependent. Different types of chlorophyll deficient mutants including albinos appeared in R₂ plant progeny where gene mutation frequency was the highest (52.4 %). The results revealed that a high frequency of somaclonal variation is possible to generate by tissue culture techniques. This revised version was published online in July 2006 with corrections to the Cover Date.     

Contrasting physiological responses by cultivars of Oryza sativa and O. glaberrima to prolonged submergence

Background and Aims Oryza glaberrima

is widely grown in flood-prone areas of African river basins and is subject to prolonged periods of annual submergence. The effects of submergence on shoot elongation, shoot biomass, leaf area and CO₂ uptake were studied and compared with those of O. sativa.

Methods

A wide selection of lines of O. sativa and O. glaberrima, including some classified as submergence tolerant, were compared in field and pot experiments. Plants were submerged completely for 31 d in a field experiment, and partially or completely for 37 d in a pot experiment in a growth chamber.

Key Results

Leaf elongation and growth in shoot biomass during complete submergence in the field were significantly greater in O. glaberrima than in O. sativa. So-called submergence-tolerant cultivars of O. sativa were unable to survive prolonged complete submergence for 31–37 d. This indicates that the mechanism of suppressed leaf elongation that confers increased survival of short-term submergence is inadequate for surviving long periods underwater. The O. sativa deepwater cultivar ‘Nylon’ and the ‘Yélé1A’ cultivar of O. glaberrima succeeded in emerging above the floodwater. This resulted in greatly increased shoot length, shoot biomass and leaf area, in association with an increased net assimilation rate compared with the lowland-adapted O. sativa ‘Banjoulou’.

Conclusions

The superior tolerance of deepwater O. sativa and O. glaberrima genotypes to prolonged complete submergence appears to be due to their greater photosynthetic capacity developed by leaves newly emerged above the floodwater. Vigorous upward leaf elongation during prolonged submergence is therefore critical for ensuring shoot emergence from water, leaf area extension above the water surface and a subsequent strong increase in shoot biomass.Key words: Flooding, leaf area, net assimilation rate, Oryza glaberrima, O. sativa, photosynthesis, rice, stress adaptation, submergence escape     

Identification and characterization of Australian wild rice strains of Oryza meridionalis and Oryza rufipogon by SINE insertion polymorphism

Of the rice species with an AA genome, Oryza meridionalis has been identified in northern Australia as a species of the annual type, among those previously classified as Oryza perennis, Oryza rufipogon or Oryza nivara. This notion has, however, led to some confusion to determine which strains belong to O. meridionalis and how different these strains are from the O. rufipogon strains of the annual type. In this paper, we examined Australian wild rice strains for the presence or absence of p-SINE1 members, which have been used for identification of the strains of species with the AA genome, by PCR using primers that hybridize to the sequences flanking each p-SINE1 member. The rice strains examined include perennial and annual strains, which have previously been described as O. rufipogon. We found that all the annual strains and other strains, whose types have not been determined, have p-SINE1 members that are specifically present at the corresponding loci in the standard strains of O. meridionalis, but do not have those which are specifically present at the corresponding loci in the strains of the other species with the AA genome. The perennial strains, however, have p-SINE1 members that are specifically present at the corresponding loci in the standard O. rufipogon strains of either the annual or the perennial type, but do not have those which are specifically present at the corresponding loci in the strains of the other species with the AA genome, including O. meridionalis. These findings support the previous notion that O. meridionalis consists of the annual strains and is a distinct species from O. rufipogon. The p-SINE1 members used in this study appear to be very useful for classification of any wild rice strains of the AA-genome species, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.     

Construction of Oryza sativa genome contigs by fingerprint strategy

We described the construction of BAC contigs of the genome of a indica variety of Oryza sativa.Guang Lu Ai 4. An entire representative(Sixfold coverage of rice chromosomes)and genetically stable BAC library of rice genome constructed in this lab has been systematically analysed by restriction enzyme fragmentation and polyacrylamide gel electrophoresis.And all the images thus obtained were subject to image-processing,which consisted of preliminary location of bands,cooperative tracking of lanes by correlation of adjacent bads.a precise densitometric pass,alignment at the marker bands with the standard,optional interactive editing,and normalization of the accepted bands.The contigs were generated based on the Computer Software specially designed for genome mapping.The number of contigs with 600 kb in length on average was 464.of contigs with 1000kb in length on average was 107; of contigs with 1500 kb in length on average was Construction of Oryza Sativa genome contigs.23.Therefor,all the contigs we have obtained ampunted up to 420 megabases in length.Considering the size of rice genome(430 megabased),the contigs generated in this lab have covered nearly 98% of the rice genome.We are now in the process of mapping the contigs to chromosomes.     

Phylogenetic relationships among Oryza species revealed by AFLP markers

 The genus Oryza to which cultivated rice belongs has 22 wild species. Seventy-seven accessions of 23 Oryza species, five related genera, and three outgroup taxa were fingerprinted using amplified fragment length polymorphism (AFLP). A total of 1191 polymorphic markers were obtained using five AFLP primer combinations. AFLP data were analyzed to study species relationships using different clustering algorithms, and the resulting phenograms were tested for stability and robustness. The findings suggest a common ancestry to the genus Oryza. Moreover, the results demonstrate that: (1) evolution in Oryza has followed a polyphyletic path wherein multiple lineages underwent independent divergence after separation early in the evolution from a common ancestor/pool of related taxa; (2) newly assigned genomes, GG for O. meyeriana and HHJJ for O. ridleyi complexes, are among the most diverged in the genus; (3) CCDD tetraploids have a relatively ancient origin among the Officinalis complex; (4) O. malampuzhaensis, O. indandamanica, O. alta, and O. grandiglumis are diverged enough to deserve species status; (5) O. officinalis and O. eichingeri (CC) are putative progenitors of O. minuta ^* O. malampuzhaensis and tetraploid O. punctata, respectively, (6) O. brachyantha is most diverged species in the genus. AFLP is reliable molecular technique and provides one of the most informative approaches to ascertain genetic relationships in Oryza, which may also be true for other related species/organisms. Received: 1 July 1998 / Accepted: 2 November 1998     

Genetic diversity in cultivars and landraces of Oryza sativa subsp. indica as revealed by AFLP markers.

Genetic diversity among 49 Indian accessions of rice (Oryza sativa subsp. indica), including 29 landraces from Jeypore, 12 modern cultivars, and 8 traditional cultivars from Tamil Nadu, was investigated using AFLP markers. In total, nine primer combinations revealed 664 AFLPs, 408 of which were found to be polymorphic. The percentage of polymorphic AFLPs was approximately the same within the cultivars and landraces. Similar results were obtained when genetic diversity values were estimated using the Shannon-Weiner index of diversity. Genetic diversity was slightly higher in the modern cultivars than in the traditional cultivars from Tamil Nadu. Among the landraces from Jeypore, the lowland landraces showed the highest diversity. The present study showed that the process of breeding modern cultivars did not appear to cause significant genetic erosion in rice. Cluster analysis and the first component of principle component analysis (PCA) both showed a clear demarcation between the cultivars and landraces as separate groups, although the genetic distance between them was narrow. The modern cultivars were positioned between the landraces from Jeypore and the traditional cultivars from Tamil Nadu. The second component of PCA further separated medium and upland landraces from lowland landraces, with the lowland landraces found closest to the traditional and modern cultivars.     

Development and mapping of Oryza glumaepatula-derived microsatellite markers in the interspecific cross Oryza glumaepatula x O. sativa

Wild germplasm of domesticated crops is a source of genetic variation little utilized in breeding programs. Interspecific crosses can potentially uncover novel gene combinations that can be important for quantitative trait analysis. The combined use of wide crosses and genetic maps of chromosomal regions associated with quantitative traits can be used to broaden the genetic basis of rice breeding programs. Oryza glumaepatula is a diploid (AA genome) wild rice species native from South and Central America. A genetic map was constructed with 162 PCR-based markers (155 microsatellite and 7 STS markers) using a backcross population derived from the cross O. glumaepatula, accession RS-16 from the Brazilian Amazon Region x O. sativa BG-90-2, an elite rice inbred line. The map included 47 new SSR markers developed from an O. glumaepatula genomic library enriched for AG/TC sequences. All SSR markers were able to amplify the O. sativa genome, indicating a high degree of SSR flanking region conservation between O. glumaepatula and O. sativa species. The map covered 1500.4 cM, with an average of one marker every 10 cM. Despite some chromosomes being more densely mapped, the overall coverage was similar to other maps developed for rice. The advantage to construct a SSR-based map is to permit the combination of the speed of the PCR reaction, and the codominant nature of the SSR marker, facilitating the QTL analysis and marker assisted selection for rice breeding programs.     

Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv. to rice,Oryza sativa L.

Oryza latifolia, a tetraploid wild relative of cultivated rice is an important source of resistance to bacterial blight (BB), the brown planthopper (BPH) and the whitebacked planthopper (WBPH). Interspecific hybrids were obtained between an elite breeding line (IR31917-45-3-2) of Oryza sativa (2n=24 AA) and O. latifolia Acc. No. 100914 (2n=48 CCDD). The crossability in F1 was 7.58% and it ranged from 0.11 to 0.62 in backcross generations. The F1 hybrid showed 2-6 II, 0-2 III, 0-1 IV and 22-32 I; the mean being 3.92 II + 0.11 III + 0.02 IV + 27.30 I per cell at diakinesis. Monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. latifolia were characterized based on morphology and isozyme banding pattern. The MAALs were designated as MAAL-1, MAAL-2, MAAL-4, MAAL-5, MAAL-6, MAAL-7, MAAL-8, MAAL-9, MAAL-10, MAAL-11 and MAAL-12. The female transmission rates of the alien chromosome varied from 4.4 to 35.5%, whereas 8 of the 11 MAALs transmitted the alien chromosome through the male gamete, the range being 1.7% (MAAL 10) to 11.9% (MAAL 12). Disomic progenies in BC3 and BC4 generations had complete resemblance to the O. sativa parent. Of the 2,295 disomic BC3F3 progenies, 309 showed introgression for resistance to BPH and 188 each for WBPH and BB resistance. Four plant progenies which were resistant to both BPH and WBPH were also resistant to BB race 2 of the Philippines. Nine of the 34 BC3F1 plants showed introgression for ten allozymes of O. latifolia, such as Est5, Amp1, Pgi1, Mdh3, Pgi2, Amp3, Pgd2, Est9, Amp2 and Sdh1, located on 8 of the 12 chromosomes. Alien introgression was also detected for morphological traits such as long awns, earliness, black hull, purple stigma and apiculus. Abnormal plants with many wild-species traits suddenly appeared in normal disomic progenies. These plants showing instability and abnormal segregation behaviour are being investigated for the activation of transposons.     

Genetic structure of Oryza rufipogon Griff. in China

Oryza rufipogon Griff. (common wild rice; CWR) is the ancestor of Asian cultivated rice (Oryza sativa L.). Investigation of the genetic structure and diversity of CWR in China will provide information about the origin of cultivated rice and the grain quality and yield. In this study, we used 36 simple sequence repeat (SSR) markers to assay 889 accessions, which were highly representative of whole germplasm in China. The analysis revealed a hierarchical genetic structure within CWR. First, CWR has diverged into two ecotypic populations, a south subtropical population (SSP) and a middle subtropical population (MSP), probably owing to natural selection by the different climates. The distribution of specific alleles and haplotypes indicated that Chinese CWR had both indica-like and japonica-like variations; the SSP was an indica-like type, whereas the MSP was more japonica-like. The SSP and MSP further diverged into five (HN, GD-GX1, GX2, FJ and YN) and two (JX-HuN1 and HuN2) geographical populations, respectively. The genetic data suggest the isolation by distance, although water systems also appear to play an important role in the formation of homogenous populations, and occasionally landscape was also involved. The population GD-GX1, which grew widely in Guangdong and Guangxi provinces, was the largest geographical population in China. It had a high level of genetic diversity (GD) and the closest genetic relationship with other inferred populations. The population HN, with the smallest SSR molecular weights and the highest level of GD, may be the most ancestral population.     

Phylogenetic analysis of Oryza rufipogon strains and their relations to Oryza sativa strains by insertion polymorphism of rice SINEs

Oryza rufipogon, the progenitor of the cultivated rice species Oryza sativa, is known by its wide intraspecific variation. In this study, we performed phylogenetic analyses of O. rufipogon strains and their relationships to O. sativa strains by using 26 newly identified p-SINE1 members from O. rufipogon strains, in addition to 23 members previously identified from O. sativa strains. A total of 103 strains of O. rufipogon and O. sativa were examined for the presence and absence of each of the p-SINE1 members at respective loci by PCR with a pair of primers that hybridize to the regions flanking each p-SINE1 member. A phylogenetic tree constructed on the basis of the insertion polymorphism of p-SINE1 members showed that O. rufipogon and O. sativa strains are classified into three groups. The first group consisted of O. rufipogon perennial strains mostly from China and O. sativa ssp. japonica strains, which included javanica strains forming a distinct subgroup. The second group consisted of almost all the O. rufipogon annual strains, a few O. rufipogon perennial strains and O. sativa ssp. indica strains. These groupings, in addition to other results, support the previous notion that annual O. rufipogon originated in the O. rufipogon perennial population, and that O. sativa originated polyphyletically in the O. rufipogon populations. The third group consisted of the other perennial strains and intermediate-type strains of O. rufipogon, in which the intermediate-type strains are most closely related to a hypothetical ancestor with no p-SINE1 members at the respective loci and to those belonging to the other rice species with the AA genome. This suggests that O. rufipogon perennial strains are likely to have originated from the O. rufipogon intermediate-ecotype population.     

Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.