FF Genome Research Articles

Cover

Panicles of wild Oryza (AA, BB, BBCC, CC, CCDD, EE and FF genomes), showing variations in size, length and branching pattern. Variations in the number of grains on a panicle may be due to differences in the reproductive strategies among the genus and species (This issue, p. 291–298).

Dual-color oligo-FISH can reveal chromosomal variations and evolution in Oryza species.

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo-FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo-FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25717 (green) and 25215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual-color oligo-FISH was used to characterize diverse chromosomal abnormalities. Oligo-FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo-FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.

Evolutionary trajectory of phytoalexin biosynthetic gene clusters in rice.

Plants frequently possess operon-like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane-related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate or syn-copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical-modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane-related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon-like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.

Occurrence and characterization of a severe isolate of Watermelon mosaic virus from Argentina

More than 50 viruses have been reported in cucurbit crops worldwide. In Argentina, cucurbit viruses have been associated with important yield losses. The most prevalent and widespread potyvirus is Watermelon mosaic virus (WMV). WMV was detected in Argentina in all cucurbit species with high incidence. In this study, a WMV isolate (WMV 1 SDE FF) was obtained from a naturally infected squash associated with a severe outbreak on melon and squash crops in an important cucurbit growing area in Santiago del Estero province (Argentina), during a survey conducted in November 2012. The fully sequenced WMV 1 SDE FF genome consists of 10,027 nucleotides and shares 96 % nt identity and 98 % aa identity with the French isolates JF273464.1|C07–014 and EU660581.1|FMF00-LL1 of the WMV molecular group 3. Using the recombination detection program RDP4, two statistically significant recombination events were identified: event 1, an 830-nt long recombinant fragment in the putative P1 coding region, and event 2, a 4071-nt recombinant fragment detected across the HC Pro, P3 and CI coding regions. The putative parental sequences detected for event 1 were the EU660586.1| FBR04–37 (major parent) and JF273468.1|C07–284 (minor parent), both from France. Putative parental sequences for event 2 were JX079685.1| WMV-ShanXi (major parent) and HQ384216.1|Dendrobium (minor parent), from China and USA, respectively. To our knowledge, this is the first complete genome of an Argentine WMV isolate. Our results provide evidence that WMV 1 SDE FF is the causal agent of the strong outbreak reported in melon and squash fields in recent years.

Fifteen Million Years of Evolution in the Oryza Genus Shows Extensive Gene Family Expansion

In analyzing gene families in the whole-genome sequences available for O. sativa (AA), O. glaberrima (AA), and O. brachyantha (FF), we observed large size expansions in the AA genomes compared to FF genomes for the super-families F-box and NB-ARC, and five additional families: the Aspartic proteases, BTB/POZ proteins (BTB), Glutaredoxins, Trypsin α-amylase inhibitor proteins, and Zf-Dof proteins. Their evolutionary dynamic was investigated to understand how and why such important size variations are observed between these closely related species. We show that expansions resulted from both amplification, largely by tandem duplications, and contraction by gene losses. For the F-box and NB-ARC gene families, the genes conserved in all species were under strong purifying selection while expanded orthologous genes were under more relaxed purifying selection. In F-box, NB-ARC, and BTB, the expanded groups were enriched in genes with little evidence of expression, in comparison with conserved groups. We also detected 87 loci under positive selection in the expanded groups. These results show that most of the duplicated copies in the expanded groups evolve neutrally after duplication because of functional redundancy but a fraction of these genes were preserved following neofunctionalization. Hence, the lineage-specific expansions observed between Oryza species were partly driven by directional selection.

Identification and diversity of functional centromere satellites in the wild rice species Oryza brachyantha

The centromere is a key chromosomal component for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, allowing each sister chromatid to faithfully segregate to each daughter cell during cell division. It is not clear what types of sequences act as functional centromeres and how centromere sequences are organized in Oryza brachyantha, an FF genome species. In this study, we found that the three classes of centromere-specific CentO-F satellites (CentO-F1, CentO-F2, and CentOF3) in O. brachyantha share no homology with the CentO satellites in Oryza sativa. The three classes of CentO-F satellites are all located within the chromosomal regions to which the spindle fibers attach and are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons, CRR-F, in the O. brachyantha centromeres. Although these CentO-F satellites are quantitatively variable among 12 O. brachyantha centromeres, immunostaining with an antibody specific to CENH3 indicates that they are colocated with CENH3 in functional centromere regions. Our results demonstrate that the three classes of CentO-F satellites may be the major components of functional centromeres in O. brachyantha.

The species of the genus Oryza and transfer of useful genes from wild species into cultivated rice, O. sativa

The genus Oryza has 24 species out of which two are cultivated (O. sativa and O. glaberrima) and 22 are wild species. Of the 22 wild species, six are in the primary gene pool of O. sativa complex and these wild species are easily crossable with the major cultivated species. These have the same AA genome as O. sativa. However, there are 10 wild species under O. officinalis complex having BB, CC, BBCC, CCDD, EE and FF genomes. The wild species of this complex are in the secondary gene pool and are cross incompatible with O. sativa. There are six most distantly related wild species with either diploids or tetraploids of GG, HHJJ and HHKK genomes and are highly cross incompatible with O. sativa. All the 22 wild species of Oryza are a vast reservoir of genes for biotic and abiotic stresses resistance. Some of the yield enhancing traits/genes from AA genome wild species have been identified and mapped with molecular markers for their integration into O. sativa genome. A broad-spectrum resistance gene for bacterial blight resistance (Xa21) has been identified in O. longistaminata and introduced into many rice cultivars. Advances in biotechnology have facilitated the development of interspecific hybrids between O. sativa and wild species of secondary and tertiary gene pools. Some important genes Pi40 and Bph18 for resistance to blast and brown planthopper, respectively, have been successfully transferred into elite cultivars from O. australiensis and the function of one blast resistance gene (Pi9) derived from O. minuta is elucidated. Many important genes from the most distantly related wild species such as O. alta, O. granulata, O. longiglumis and O. coarctata are expected to be transferred into cultivated rice in the future using the latest tools of molecular genetics and biotechnology.

New insights into Oryza genome evolution: high gene colinearity and differential retrotransposon amplification

An approximately 247-kb genomic region from FF genome of wild rice Oryza brachyantha, possessing the smallest Oryza genome, was compared to the orthologous approximately 450-kb region from AA genome, O. sativa L. ssp. japonica. 37 of 38 genes in the orthologous regions are shared between japonica and O. brachyantha. Analyses of nucleotide substitution in coding regions suggest the two genomes diverged approximately 10 million years ago. Comparisons of transposable elements (TEs) reveal that the density of DNA TEs in O. brachyantha is comparable to O. sativa; however, the density of RNA TEs is dramatically lower. The genomic fraction of RNA TEs in japonica is two times greater than in O. brachyantha. Differences, particularly in RNA TEs, in this region and in BAC end sequences from five wild and two cultivated Oryza species explain major genome size differences between sativa and brachyantha. Gene expression analyses of three ObDREB1 genes in the sequenced region indicate orthologous genes retain similar expression patterns following cold stress. Our results demonstrate that size and number of RNA TEs play a major role in genomic differentiation and evolution in Oryza. Additionally, distantly related O. brachyantha shares colinearity with O. sativa, offering opportunities to use comparative genomics to explore the genetic diversity of wild species to improve cultivated rice.

Isolation and characterization of the centromeric BAC clones from differnet genomes in genus Oryza

Centromeres play an important role in ensuring the correct segregation and transmission of chromosome during mitosis and meiosis in eukaryotes. In this research, we constructed five BAC libraries for diploid wild rice with different genomes. Together with the technique of colony blot hybridization and fluorescence in situ hybridization (FISH), centromere-related BAC clones were screened and characterized from different genomes. Meanwhile, co-hybridization was detected between these clones and the five genomes. The results from this study demonstrated that: (1) there were centromere-specific satellite repeat in Oryza officinalis (CC genome) and O. brachyantha (FF genome), respectively, and centromere-specific CRR-related sequence was found in O. brachyantha; (2) homology sequences of CentO and CRR of O. sativa (AA genome) were detected on all centromeres of O. glaberrima (AA genome), O. punctata (BB genome) and O. australiensis (EE genome); And (3) the two somatic chromosomes of O. officinalis comprised of homology sequences of CentO satellites as revealed FISH analysis probed with RCS2. Homology sequences of CRR of O. sativa were also detected on all centromeres of O. officinalis. The results provided a foundation toward cloning the centromeric sequences from different genomes of genus Oryza, studying centromere organization and evolution of different genome, analyzing the relationship between centromeric structure and function among different genome.

Chromatin immunoprecipitation cloning reveals rapid evolutionary patterns of centromeric DNA in Oryza species

The functional centromeres of rice (Oryza sativa, AA genome) chromosomes contain two key DNA components: the CRR centromeric retrotransposons and a 155-bp satellite repeat, CentO. However, several wild Oryza species lack the CentO repeat. We developed a chromatin immunoprecipitation-based technique to clone DNA fragments derived from chromatin containing the centromeric histone H3 variant CenH3. Chromatin immunoprecipitation cloning was carried out in the CentO-less species Oryza rhizomatis (CC genome) and Oryza brachyantha (FF genome). Three previously uncharacterized genome-specific satellite repeats, CentO-C1, CentO-C2, and CentO-F, were discovered in the centromeres of these two species. An 80-bp DNA region was found to be conserved in CentO-C1, CentO, and centromeric satellite repeats from maize and pearl millet, species which diverged from rice many millions of years ago. In contrast, the CentO-F repeat shows no sequence similarity to other centromeric repeats but has almost completely replaced other centromeric sequences in O. brachyantha, including the CRR-related sequences that normally constitute a significant fraction of the centromeric DNA in grass species.

Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza

Inter simple sequence repeat (ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in Oryza. Forty two genotypes including 17 wild species, representing AA,BB,CC,EE,FF,GG,BBCC,CCDD, and HHJJgenomes, two cultivated species, Oryza sativa (AA) and Oryza glaberrima (AA), and three related genera, Porteresia coarctata, Leersia and Rhynchoryza subulata, were used in ISSR analysis. A total of 30 ISSR primers were screened representing di-, tri-, tetra- and penta-nucleotide repeats, of which 11 polymorphic and informative patterns were selected to determine the genetic diversity. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered 42 genotypes according to their respective genomes. ISSR analysis suggests that the genus Oryza may have evolved following a polyphyletic pathway; Oryza brachyantha (FF genome) is the most divergent species in Oryza and Oryza australiensis (EE genome) does not fall under the Officinalis complex. DNA profiles based on ISSR markers have revealed potential diagnostic fingerprints for various species and genomes, and also for individual accessions/cultivars. Additionally ISSR revealed 87 putative genome/species-specific molecular markers for eight of the nine genomes of Oryza. The ISSR markers are thus useful in the fingerprinting of cultivated and wild species germplasm, and in understanding the evolutionary relationships of Oryza.

Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements

As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili. Phage IKe has a genome with 55% identity with the Ff genome and infects E. coli strains bearing N pili. The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3′ end and unique 5′ ends. The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs. However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3′ end. Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function. By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host endoribonucleases, the results argue that the pattern is not simply fortuitous.

A novel repetitive DNA sequence in the genus Oryza

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.

Genome specificity of rDNA spacer fragments from Oryza sativa L.

The intergenic spacer derived from a cloned rDNA unit from a cultivated rice was dissected into several subclones, which were used as probes to analyze sequence homologies between rDNA spacers from wild rice belonging to genome types AA, BB, CC, EE, FF, BBCC, and CCDD. This analysis allowed us to detect several regions with different degrees of homology. A series of 250-260 bp repeats is located in the central part of the AA spacer. This sequence cross-hybridizes with the BB, CC, BBCC, and FF genomes, but is absent in the EE and CCDD genomes. Regions proximal to 25S and 18S sequences are well conserved in all genomes. Finally, two adjacent sequences of 61 bp and 94 bp, located downstream from the repeats, have been found to have a narrow genomic specificity, restricted respectively to AA and FF genomes for the first one and to AA for the second. These data provide new information on the evolution of the ribosomal RNA gene spacer within a complex of related species, and add to our knowledge on the relationship between the various rice genomes.

DNA sequence of the filamentous bacteriophage Pf1

The genome of the class II filamentous bacteriophage Pf1 has been sequenced by a combination of the chain termination and chemical degradation methods. It consists of 7349 nucleotides in a closed, circular loop of single-stranded DNA. The size and position of its open reading frames (ORFs) in general resemble those of other filamentous bacteriophage genomes. The size and position of the spaces between the ORFs have not been conserved, however, and six short reading frames (2 of which overlap) occupy a region corresponding to that filled by genes 2 and 10 in the Ff genome. Most of the ORFs are preceded by sequences resembling ribosome binding sites from the phage's host, Pseudomonas aeruginosa, that appear to differ somewhat from their counterparts in Escherichia coli. A search for sequences related to known pseudomonad promoters suggests that the promoters in this bacteriophage may well be ntr-dependent, with the two strongest preceding the gene for the major coat protein (gene 8) and another ORF (430). Gene 8 is followed by a sequence with the properties of a ϱ-independent terminator of transcription, like that at the same position in the genome of Ff. The Pf1 genome contains no collection of potential stem-and-loop structures corresponding to those that initiate replication of Ff DNA and assembly of the Ff virion, although isolated structures of this kind are present. The available evidence suggests that at least 13 of the 14 major ORFs are expressed. Overall, the organization of the Pf1 genome differs from that of the other class II filamentous phage whose genome has been sequenced, Pf3, as much as it does from that of the class I phages Ff and IKe.

Genome-specific repetitive sequences in the genus Oryza

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.

Distribution of d-alanylglycine and related compounds in Oryza species

d-Alanine was detected abundantly in all Oryza species, the genome formula of which is known. In the strains containing the AA, BB, BBCC and CC genomes, d-alanine is distributed in the form of d-alanylglycine while in the strains containing the EE and FF genomes, it is distributed in the form of d-alanyl-d-alanine. In the strains containing the CCDD genome, d-alanylglycine or d-alanyl-d-alanine is present. Exogenously supplied d-alanine tended to be incorporated into d-alanylglycine, d-alanyl-d-alanine and d-alanyl-l-alanine in the strains of the d-alanylglycine type, and only into d-alanyl-d-alanine in those of the d-alanyl-d-alanine type.

Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe: Comparison with the genome of the F-specific filamentous phages M13, fd and f1

The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff). The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome. The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx. 55%) and that their genomes contain ten homologous genes, the order of which is identical. Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules. The extent of homology between the genes of IKe and Ff differs significantly from one gene to another. Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis. During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities. Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.