Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by analysis of nuclear RFLPs

Summary Ninety-three accessions representing 21 species from the genus Oryza were examined for restriction fragment length polymorphism. The majority (78%) of the accessions, for which five individuals were tested, were found to be monomorphic. Most of the polymorphic accessions segregated for only one or two probes and appeared to be mixed pure lines. For most of the Oryza species tested, the majority of the genetic variation (83%) was found between accessions from different species with only 17% between accessions within species. Tetraploid species were found to have, on average, nearly 50% more alleles (unique fragments) per individual than diploid species reflecting the allopolyploid nature of their genomes.Classification of Oryza species based on RFLPs matches remarkably well previous classifications based on morphology, hybridization and isozymes. In the current study, four species complexes could be identified corresponding to those proposed by Vaughan (1989): the O. ridleyi complex, the O. meyeriana complex, the O. officinalis complex and the O. sativa complex. Within the O. sativa complex, accessions of O. rufipogon from Asia (including O. nivara) and perennial forms of O. rufipogon from Australia clustered together with accessions of cultivated rice O. sativa. Surprisingly, indica and japonica (the two major subspecies of cultivated rice) showed closer affinity with different accessions of wild O. Rufipogon than to each other, supporting a hypothesis of independent domestication events for these two types of rice. Australian annual wild rice O. meridionalis (previously classified as O. rufipogon) was clearly distinct from all other O. rufipogon accessions supporting its recent reclassification as O. meridionalis (Ng et al. 1981). Using genetic relatedness as a criterion, it was possible to identify the closest living diploid relatives of the currently known tetraploid rice species. Results from these analyses suggest that BBCC tetraploids (O. malampuzhaensis, O. punctata and O. minuta) are either of independent origins or have experienced introgression from sympatric C-genome diploid rice species. CCDD tetraploid species from America (O. latifolia, O. alta and O. grandiglumis) may be of ancient origin since they show a closer affinity to each other than to any known diploid species. Their closest living diploid relatives belong to C genome (O. eichingeri) and E genome (O. Australiensis) species. Comparisons among African, Australian and Asian rice species suggest that Oryza species in Africa and Australia are of polyphyletic origin and probably migrated to these regions at different times in the past.Finally, on a practical note, the majority of probes used in this study detected polymorphism between cultivated rice and its wild relatives. Hence, RFLP markers and maps based on such markers are likely to be very useful in monitoring and aiding introgression of genes from wild rice into modern cultivars.     

Genome-specific repetitive sequences in the genus Oryza

Summary Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species divergence. In this paper, we report the isolation and characterization of four genome-type specific repetitive DNA sequences in the genus Oryza. Sequences specific to the AA, CC, EE or FF genome types are described. These genome-type specific repetitive sequences will be useful in classifying unknown species of wild or domestic rice, and in studying genome evolution at the molecular level. Using an AA genome-specific repetitive DNA sequence (pOs48) as a hybridization probe, considerable differences in its copy number were found among different varieties of Asian-cultivated rice (O. sativa) and other related species within the AA genome type. Thus, the relationship among some of the members of AA genome type can be deduced based on the degree of DNA sequence similarity of this repetitive sequence.     

Phylogenetic relationships in the orchid genus Serapias L. based on noncoding regions of the chloroplast genome

A molecular phylogenetic analysis was performed on 14 species of the Mediterranean unrewarding orchid genus Serapias using sequences of four noncoding regions of chloroplast DNA. This study has led to a new interpretation of the evolutionary relationships in this genus. The well-defined phylogenetic tree supports a division of taxa into two main clades, each including two minor groups. The molecular relationships found in this study differ from those defined by traditional systematic morphological assessments. By comparing the variation in sequence to variations in floral traits, we propose that the split in the two main lineages reflects an early differentiation of flower size, perhaps due to the shift from allo- to self-pollination. Conversely, the relationships within each minor group do not reflect floral size variation; therefore, we presume that this diversification resulted from genetic drift, local selection forces, and multiple, independent transitions towards self-pollination and polyploidy.     

Characteristics of nuclear DNA in the genus Oryza

Summary DNAs have been isolated from various Oryza species and studied using physical techniques. The percent of guanine plus cytosine has been determined by thermal denaturation. While the base composition varied between the species, no heterogeneity in the base pair distribution was observed. Renaturation kinetics data of DNAs from different species show that the proportion of repeated DNA sequences vary considerably depending on the DNA content per cell, whereas the nonrepetitive DNA component remains relatively constant. These results suggest that in addition to a small range of DNA variation between the species, changes in the base composition and proportion of repeated sequences have accompanied divergence of the species within the genus.     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Origin and phylogeny of Oryza species with the CD genome based on multiple-gene sequence data

The CD genome species in the genus Oryza are endemic to Latin America, including O. alta, O. grandiglumis and O. latifolia. Origins and phylogenetic relationship of these species have long been in dispute and are still ambiguous due to their homogeneous genome type, similar morphological characteristics and overlapping distribution. In the present study, we sequenced two chloroplast fragments (matK and trnL-trnF) and portions of three nuclear genes (Adh1, Adh2 and GPA1) from sixteen accessions representing seven species with the C, CD, and E genomes, as well as one G genome species as the outgroup. Phylogenetic analyses using parsimony and distance methods strongly supported that the CD genome originated from a single hybridization event, and that the C genome species (O. officinalis or O. rhizomatis instead of O. eichingeri) served as the maternal parent while the E genome species (O. australiensis) was the paternal donor during the formation of CD genome. In addition, the consistent phylogenetic relationships among the CCDD species indicated that significant divergence existed between O. latifolia and the other two (O. alta and O. grandiglumis), which corroborated the suggestion of treating the latter two as a single species or as taxa within species.We thank Tao Sang of Michigan State University (East Lansing, USA) and Bao-rong Lu of Fudan University (Shanghai, China) for their encouragement and assistance. We are also grateful to the International Rice Research Institute (Manila, Philippines) for providing plant material for this study. This research was supported by the Chinese Academy of Sciences (kscxz-sw-101A), the National Natural Science Foundation of China (30025005) and the Program for Key International S & T Cooperation Project of P. R. China (2001CB711103).     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Aminoacid profiles in the study of phylogenetic relationships in the genusOryza

Summary The amino acid profiles in seeds of thirteen different species ofOryza, including two cultivated rices,O. glaberrima andO. sativa and the two major geographical racesindica andjaponica were studied using an automatic amino acid analyser to assess differences in the profiles of cultivated species and their wild progenitors. The polygon graphic method was employed to envision the species relationship. Essential amino acid profiles in different species were also compared with those of the Food and Agriculture Organization (FAO) standards. The results suggest a wide range of variability amongOryza species for lysine (up to 4.4% as against 3.5% in cultivated rices) and other essential amino acids. This will be of considerable interest to rice breeders, when after overcoming genetic barriers, the possible utilization of these species in rice breeding becomes feasible.     

Homologous recombination mediated by two palindromic repeated sequences in the mitochondrial genome of Oryza

Palindromic repeated sequences (PRSs) are distributed in at least ten regions of the mitochondrial (mt) genome of rice and are, apparently, mobile. In the present study, we examined the possibility of homologous recombination via some PRSs during the course of evolution of Oryza. We first performed Southern hybridization of the DNA from 11 species (18 strains) of Oryza in order to identify the distribution of PRSs in the mitochondrial genome of Oryza. The hybridization patterns revealed genome type-specific and/or species-specific variations. We speculated that homologous recombination via some PRSs might have made a contribution to such variations. After subsequent polymerase chain reaction, Southern hybridization and sequencing, we concluded that homologous recombination mediated by two PRSs occurred in the mtDNA of Oryza after divergence of the BB genome type and the other genome types of Oryza. Evidence was obtained that some PRSs were involved in both insertion and recombination events during the evolution of Oryza. Our results indicate, therefore, that PRSs have contributed considerably to the polymorphism of Oryza mtDNAs.     

Molecular taxonomic relationships in the genus Vigna based on RFLP analysis

Summary The taxonomy of the genus Vigna has been primarily based on morphological attributes. We have used 27 genomic clones from soybean, common bean, mungbean and cowpea to examine restriction fragment length polymorphism (RFLP) among 44 accessions of different species belonging to four subgenera of the genus Vigna. One accession each of common bean (Phaseolus vulgaris) and soybean (Glycine max) was included in the study. Total DNA from the various genotypes was digested with one restriction enzyme (Eco RV). Results of a numerical taxonomic analysis showed a high level of genetic variation within the genus with a remarkably higher amount of variation associated with Vigna sp. from Africa relative to those from Asia. The distinctness of the Asiatic grams in subgenus Ceratotropis, cowpea in section Catiang, bambara groundnut (V. subterranean) and members of the subgenus Plectotropis was elucidated by this study. Members of the subgenus Plectotropis were closer in genome homology to those of subgenus Vigna section Catiang than to those of subgenus Ceratotropis. The relative positions of some genotypes to one another on the dendrogram and minimum spanning tree were discussed in regard to hybridisations aimed generating well-saturated genomic maps and interspecies transfer of desirable genes.     

Diversity in the Integrase Coding Domain of a gypsy-like Retrotransposon Among Wild Relatives of Rice in the Oryza officinalis Complex

The Oryza officinalis complex is a genetically diverse, tertiary genepool of rice. We analyzed part of the primary structure of the integrase coding domain (ICD) of a gypsy-like retrotransposon from species of the O. officinalis species complex. PCR was performed with degenerate primers that hybridized to conserved sequences in the integrase genes of gypsy-type retrotransposons, using total DNA from different species of the O. officinalis complex as templates. Cloning and sequencing of the PCR products showed that the amplified fragments are highly homologous to each other (75–90%) and belong to one family of retrotransposons that is related to the previously studied RIRE-2 element from rice. Two main subfamilies of 292 and 351 bp were distinguished. Analysis of primary sequence data supports previous reports that sequence divergence during vertical transmission has been the major influence on the evolution of gypsy-type retrotransposons in Oryza species. Based on sequence data phylogenetic relationships among species of the O. officinalis complex were estimated. The data suggests that O. eichingeri is more closely related to the ancestral species of the complex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Polysomes and intracisternal accumulations in enucleate sieve elements of rice (Oryza sativa L.)

Polysomes in sieve elements of rice (Oryza sativa L.) were studied with the electron microscope. The polysomes were found on the rough endoplasmic reticulum (ER) present in immature sieve elements and also on the cisternae of aggregated ER in the parietal layer of mature, enucleate sieve elements. In the immature sieve elements the ER cisternae existed as narrow profiles while in the mature sieve elements the ER cisternae were considerably dilated and contained a fibrillar material and, occasionally, electron-opaque inclusions. In addition to the aggregated ER, single profiles of ER were found applied to the lateral walls and also the sieve plates. These cisternae also bore ribosomes and were separated from the plasmalemma by a narrow, dense space. In the mature sieve elements much of the surface of the ER membranes was covered with polysomes. The dimensions of the polysomes are described and the possibility that they contribute to the formation of the fibrillar material in the intracisternal space is discussed.Abbreviations ER endoplasmic reticulum     

A comprehensive expression analysis of the starch synthase gene family in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the roles of the isogenes encoding starch synthase (EC 2.4.1.21) in rice (Oryza sativa L.), a comprehensive expression analysis of the gene family was conducted. Extensive searches for starch synthase genes were done in the databases of both the whole genome and full-length cDNAs of rice, and ten genes were revealed to comprise the starch synthase gene family. Multi-sequence alignment analysis of the starch synthase proteins from rice and other plant species suggested that they were grouped into five classes, soluble starch synthase I (SSI), SSII, SSIII, SSIV and granule-bound starch synthase (GBSS). In rice, there was one gene for SSI, three for SSII and two each for SSIII, IV and GBSS. The expression pattern of the ten genes in the developing caryopsis was examined by semi-quantitative RT–PCR analysis. Based on the temporal expression patterns, the ten genes could be divided into three groups: (i) early expressers (SSII-2, III-1, GBSSII), which are expressed in the early stage of grain filling; (ii) late expressers (SSII-3, III-2, GBSSI), which are expressed in the mid to later stage of grain filling; and (iii) steady expressers (SSI, II-1, IV-1, IV-2), which are expressed relatively constantly during grain filling. Within a caryopsis, the three gene groups spatially share their expression, i.e. early expressers in the pericarp, the late expressers in the endosperm and the steady expressers in both tissues. In addition, this grouping was reflected in the expression pattern of various rice tissues: expression in non-endosperm, endosperm or all tissues examined. The implications in this spatio-temporal work sharing of starch synthesis isogenes are discussed.Abbreviations DAF Days after flowering - GBSS Granule-bound starch synthase - SS Soluble starch synthase     

Allele mining for stress tolerance genes in Oryza species and related germplasm

Allele mining exploits the deoxyribonucleic acid (DNA) sequence of one genotype to isolate useful alleles from related genotypes. The international project to sequence the genome of Oryza sativa L cv. Nipponbare will make allele mining possible for all genes of rice and possibly related cereals. We used a rice calmodulin gene, a rice gene encoding a late embryogenesis-associated protein, and salt-inducible rice gene to optimize the polymerase chain reaction (PCR) for allele mining of stress tolerance genes on identified accessions of rice and related germplasm. Two sets of PCR primers were designed for each gene. Primers based on the 5′ and 3′ untranslated region of genes were found to be sufficiently conserved so as to be effective over the entire range of germplasm in rice for which the concept of allelism is applicable. However, the primers based on the adjacent amino (N) and carboxy (C) termini amplify additional loci.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.)

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6−phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Effects of submergence on development and gravitropism in the coleoptile of Oryza sativa L.

Caryopses of rice (Oryza sativa L. cv. Sasanishiki) were germinated in air or under water. In submerged seedlings a twofold increase in coleoptile growth rate and an inhibition of root growth was observed. The amount of starch in the amyloplasts of submerged coleoptiles was substantially reduced compared to the air-grown control plants and plastids had a proplastidic character. During the rapid elongation of coleoptiles under water, the osmotic concentration of the press sap remained constant, whereas in air-grown coleoptiles a decrease was measured. Determination of curvature of gravistimulated air-grown and submerged shoots was carried out by placing the coleoptiles horizontally in air of 98% relative humidity. Air-grown coleoptiles reached a vertical orientation within 5 h after onset of gravistimulation. In coleoptiles germinated under water the first signs of consistent negative gravitropic bending occurred after 4–5 h and curvature was complete after 24 h. During the first 5 h of gravistimulation the water-grown coleoptiles grew at an average rate of 0.39 mm·h^–1, whereas in air-grown coleoptiles a rate of 0.27 mm·h^–1 was measured. Concomitant with the delayed onset of gravitropic bending of the water-grown coleoptiles, a change in plastid ultrastructure and an increase in starch content was observed. We conclude that the gravitropic responsiveness of the rice coleoptile depends on the presence of starch-filled amyloplasts.We wish to thank H.-J. Ensikat for technical assistance with the scanning electron microscopy. Supported by the Bundesminister für Forschung und Technologie and the Deutsche Forschungsgemeinschaft.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.