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.     

Genetic analyses of Oryza species by molecular markers for chloroplast genomes

Summary Relationships in a wide range of Oryza species (13 species) were analyzed using the large subunits (LS) of Fraction I protein (Rubisco) and the Bam HI restriction patterns of chloroplast DNA (ctDNA) as molecular markers. Four types of LS were detected by isoelectrofocusing with and without S-carboxymethylation. The close relation between AA and CCDD genome species was suggested by analyses of LS and ctDNA. Intraspecific variation in O. latifolia was detected at the levels of both LS and ctDNA. The LS of the BB, BBCC, and CC genomes and FF (O. brachyantha) were not distinguishable, although the native Rubisco of the latter was slightly different from those of the first three. It was also shown that O. australiensis, the only EE genome species, might have evolved differently than the other Oryza species.     

Allele mining in crops: Prospects and potentials

Enormous sequence information is available in public databases as a result of sequencing of diverse crop genomes. It is important to use this genomic information for the identification and isolation of novel and superior alleles of agronomically important genes from crop gene pools to suitably deploy for the development of improved cultivars. Allele mining is a promising approach to dissect naturally occurring allelic variation at candidate genes controlling key agronomic traits which has potential applications in crop improvement programs. It helps in tracing the evolution of alleles, identification of new haplotypes and development of allele-specific markers for use in marker-assisted selection. Realizing the immense potential of allele mining, concerted allele mining efforts are underway in many international crop research institutes. This review examines the concepts, approaches and applications of allele mining along with the challenges associated while emphasizing the need for more refined ‘mining’ strategies for accelerating the process of allele discovery and its utilization in molecular breeding.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

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.     

Assessment of genome relationships in the genus Oryza L. based on seed-protein profile analysis

Summary Cultivated and wild Oryza species belonging to different genomic groups were studied with regard to their soluble seed-protein profiles. There is an essential uniformity in the banding patterns within various genomes and the basic patterns are not species-specific but genome-specific. O. meridionalis contains a subgenome similar to the A genome of O. rufipogon. Certain specific bands present among A genome species have been found to be useful in tracing the phylogenetic affinity between the cultivated species and their presumed wild progenitors.     

Chloroplast DNA diversity in wild and cultivated species of rice (Genus Oryza,section Oryza). Cladistic-mutation and genetic-distance analysis

Summary Using a novel nonaqueous procedure, chloroplast DNA was isolated from 318 individual adult rice plants, representing 247 accessions and the breadth of the diversity in section Oryza of genus Oryza. Among them, 32 different cpDNA restriction patterns were distinguished using the restriction endonucleases EcoRI and AvaI, and they were further characterized by restriction with BamHI, HindIII, SmaI, PstI, and BstEII enzymes. The differences in the electrophoretic band patterns were parsimoniously interpreted as being the result of 110 mutations, including 47 restriction site mutations. The relationships between band patterns were studied by a cladistic analysis based on shared mutations and by the computation of genetic distances based on shared bands. The deduced relationships were compared with earlier taxonomical studies. The maternal parents for BC genome allotetraploids were deduced. Within species, cpDNA diversity was found larger in those species with an evolutionary history of recent introgression and/or allotetraploidization. Occasional paternal inheritance and recombination of cpDNA in rice was suggested.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

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.     

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.     

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.     

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).     

Comparative spikelet morphology ofOryza schlechteri Pilger and related species ofLeersia andOryza (Poaceae)

Oryza schlechteri has only recently been rediscovered in Papua New Guinea. This species is quite distinct from otherOryza species but resembles closely related genusLeersia in its small panicle and profusely spreading habit. On the basis of spikelet morphology that has previously been used to distinguishLeersia andOryza species,O. schlechteri was compared withO. brachyantha. O. longiglumis, O. sativa, L. perrieri, L. tisseranti, andL. hexandra to clarify its generic position. Results corroborate that the presence of a sterile lemma and a striated spikelet epidermal (abaxial) surface lacking siliceous triads inO. schlechteri allies this species with otherOryza species rather than withLeersia.     

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.     

Shoot regeneration capacity from roots and transgenic hairy roots of tomato cultivars and wild related species

The organogenetic competence of roots and Agrobacterium rhizogenes-induced hairy roots of twelve Lycopersicon genotypes was investigated. Both roots and hairy roots of L. peruvianum, L. chilense, L. hirsutum and two L. peruvianum-derived genotypes regenerated shoots after 2–4 weeks of incubation on zeatin-contained medium. Anatomical analysis showed that shoot regeneration in roots could be direct or indirect, depending on the genotype considered. Hairy roots showed considerable differences in their morphogenetic responses, when compared to the corresponding non-transgenic roots. The differences observed may reflect the influence of the introduced rol genes on hormonal metabolism/sensitivity. Hairy root-derived T0 plants had shortened internodes, wrinkled leaves and abundant root initiation, and most produced flowers and fruits with viable seeds. The hairy root syndrome was detected early in germinating T1 seedlings as a strong reduction in the hypocotyl length. Our data point to the possibility of the use of A. rhizogenes, combined with regenerating Lycopersicon genotypes, in a very simple protocol, based on genetic capacity instead of special procedures for regeneration, to produce transgenic tomato plants expressing rol genes, as well as, genes present in binary vectors. Furthermore, the regeneration differences observed in each Lycopersicon genotype and in transgenic materials expressing rol genes open the possibility for their use in the analysis of both the biochemical and the genetic background of organogenetic competence. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular approaches for identification and construction of novel insecticidal genes for crop protection

Summary The insecticidal cry (crystal) genes from Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. Discovery of new insecticidal genes is of importance for delaying the development of resistance in target insects. The diversity of Bt strains facilitates isolation of new types of cry and vip (vegetative insecticidal protein) genes. PCR is a useful technique for quick and simultaneous screening of Bt strains for classification and prediction of insecticidal activities. PCR together with other methods of analysis such as RFLP, gene sequence determination, electrophoretic, immunological and chromatographic analysis of Cry proteins and insect bioassays for evaluation of toxicity have been employed for identification of new insecticidal proteins. Some other new approaches have also been devised. Many Bt strains with novel insecticidal genes have been found. A desired combination of Cry proteins can be assembled via site-specific recombination vectors into a recipient Bt strain to create a genetically improved biopesticide. For better pest control, the cry genes have been transferred to plants. Stacking of more than one insecticidal gene is required for resistance management in transgenic crops. Modification of Cry proteins through protein engineering for increasing the toxicity and/or the insecticidal spectrum is also a promising approach, but requires detailed understanding of the structure and function of these proteins and analysis of toxin-receptor interactions. More research into this area will provide useful insights for the design of toxins for management of insect resistance. Insecticidal genes from other bacteria and plants are also being examined for their potential for deployment in transgenic crops. Stringent implementation of resistance management is needed for maintaining the efficacy of Bt transgenic crops and deriving maximum economic and environmental benefit.     

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.