Physical mapping of rDNA and heterochromatin in chromosomes of 16 Coffea species: a revised view of species differentiation.

The chromosome organization among 15 wild diploid Coffea species and cultivated tetraploid C. arabica was determined by fluorochrome banding (CMA, DAPI) and double fluorescence in-situ hybridization (FISH) of 5S and 18S rDNA achieved on the same chromosome plates. Two to five chromosome pairs (plus one putative chromosome B) are marked. Overall, there are two SAT-chromosome pairs for East African species and one for the Malagasy and the West and Central African species. 18S rDNA loci are telomeric and strongly marked the SAT-chromosome pairs. Generally, only one pericentromeric 5S rDNA locus characterized East African species, while an additional minor locus co-localized with the 18S rDNA-SAT locus for the Malagasy species and West and Central African species. A combination of rDNA FISH plus CMA and DAPI banding patterns enables identification of almost all the species, even those for which the genetic or botanical status is still being discussed. C. arabica clearly appears to be an allotetraploid species, including one genome from East Africa and one from West and Central Africa. However, since the minor 5S rDNA-SAT locus present in West/Central African genomes is not detected, two evolutionary hypotheses could be put forward for C. arabica. Considering only the diploid species, global trends are obvious in rDNA signal patterns, genome size variations, and geographic distribution of the species, but there are no clear evolutionary trends. However, complex interactions between these factors and environmental growing conditions exist, which have resulted in loss and gain of rDNA loci and probably also in copy repeat number variations in each rDNA family.

Expression and inheritance of multiple transgenes in rice plants.

The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation.

Near immunity to rice tungro spherical virus achieved in rice by a replicase-mediated resistance strategy.

ABSTRACT Rice tungro disease is caused by rice tungro bacilliform virus (RTBV), which is responsible for the symptoms, and rice tungro spherical virus (RTSV), which assists transmission of both viruses by leafhoppers. Transgenic japonica rice plants (Oryza sativa) were produced containing the RTSV replicase (Rep) gene in the sense or antisense orientation. Over 70% of the plants contained one to five copies of the Rep gene, with integration occurring at a single locus in most cases. Plants producing antisense sequences exhibited significant but moderate resistance to RTSV (60%); accumulation of antisense RNA was substantial, indicating that the protection was not of the homology-dependent type. Plants expressing the full-length Rep gene, as well as a truncated Rep gene, in the (+)-sense orientation were 100% resistant to RTSV even when challenged with a high level of inoculum. Accumulation of viral RNA was low, leading us to conclude that RTSV Rep-mediated resistance is not protein-mediated but is of the cosuppression type. Resistance was effective against geographically distinct RTSV isolates. In addition, RTSV-resistant transgenic rice plants were unable to assist transmission of RTBV. Such transgenic plants could be used in an epidemiological approach to combat the spread of the tungro disease.

Isozymic variability of traditional rice (Oryza sativa L.) in Africa.

Eight hundred and thirtyone traditional varieties of rice, Oryza sativa L., were collected in Africa and analysed for their isozymic variability on 15 enzymatic systems, representing 37 presumed loci. There appears to be a correlation between the type of rice growing and the two groups Indica and Japonica. The degree of genetic diversity is nearly equal in African rice and the Asian one. Alleles due to introgression or mutational events were identified. The results suggest that the evolution of O. sativa is continuous in Africa by means of inter-subspecific or inter-specific crosses.

An improved rice transformation system using the biolistic method.

Immature embryos and embryogenic calli of rice, both japonica and indica subspecies, were bombarded with tungsten particles coated with plasmid DNA that contained a gene encoding hygromycin phosphotransferase (HPH, conferring hygromycin resistance) driven by the CaMV 35S promoter or Agrobactenum tumefaciens NOS promoter. Putatively transformed cell clusters were identified from the bombarded tissues 2 weeks after selection on hygromycin B. By separating these cell clusters from each other, and by stringent selection not only at the callus growth stage but also during regeneration and plantlet growth, the overall transformation and selection efficiencies were substantially improved over those previously reported. From the most responsive cultivar used in these studies, an average of one transgenic plant was produced from 1.3 immature embryos or from 5 pieces of embryogenic calli bombarded. Integration of the introduced gene into the plant genome, and inheritance to the offspring were demonstrated. By using this procedure, we have produced several hundred transgenic plants. The procedure described here provides a simple method for improving transformation and selection efficiencies in rice and may be applicable to other monocots.

Genome size variations in diploid African Coffea species.

Flow cytometry was conducted to evaluate genome size diversity among African diploid species of the Coffea genus. The study included 15 species and six new taxa from Congolese and Cameroonian forest regions which have yet to be botanically characterized. Between-population differences were also recorded in some cases. These evaluations using an internal standard were highly correlated with previous results obtained with an external standard, but differences of up to 18 % existed for some species, involving stoichiometric errors. Consequently, genome size variation between species and within species are discussed as true genome size differences or stoichiometric errors. Environmental and phenotypic correlations with genome size are also discussed.

Distribution and organization of a tandemly repeated 352-bp sequence in the oryzae family.

A 352-bp EcoRI fragment from rice DNA was cloned and shown to be a member of a tandem repeat. Sequence determination revealed homologies with human alpha satellite DNA and maize knob heterochromatin specific repeat. This 352-bp sequence is highly specific for the AA genome of rice. However, copy number and sequence organization are variable, depending on the accession analyzed. Several examples of amplification were observed in O. rufipogon and O. longistaminata. Use of resolutive polyacrylamide gel electrophoresis and 4-bp cutter enzymes allowed one to distinguish between the Indica and Japonica subtypes of O. sativa. The same method also discriminates between two groups of O. rufipogon, the presumed ancestor of O. sativa, suggesting that the present day Indica and Japonica subtypes originated independently from two O. rufipogon distinct populations.

Cassava vein mosaic virus (CsVMV), type species for a new genus of plant double stranded DNA viruses?

The complete sequence of 8159 nucleotides of the double stranded DNA genome of cassava vein mosaic virus (CsVMV) was determined (# U59751) and revealed a significant difference in genome organization when compared with a previous report (# U20341). When transferred to cassava plants by microbombardment, the full length CsVMV clone was infectious, confirming the genome organization here described. Sequence comparisons between CsVMV and members of the genera Caulimovirus and Badnavirus revealed high homologies between consensus sequences of several proteins that are indispensable for virus replication, including a potential transactivator factor not reported previously. The presence of a sequence complementary to a plant Met tRNA confirms that CsVMV is a plant pararetrovirus and is most closely related to members of the genus Caulimovirus as previously assessed. However, differences in genome organization, number and size of the ORFs, in addition to sequence comparisons with other plant pararetroviruses, shows that either the genetic variability of caulimoviruses is much greater than previously thought, or that CsVMV is the unique representative of a new genus within the Caulimoviridae family. On the basis of this study, it is proposed to upgrade the floating genus Caulimovirus to the family level and to divide the Caulimoviridae family into at least three genera with CsVMV being the type member of a new genus.

Functional organization of the cassava vein mosaic virus (CsVMV) promoter.

Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of beta-glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.

Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter.

The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to the uidA reporter gene to test promoter expression. The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene. Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions. The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants. The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression. In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips.

Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in Cameroon.

Stem cuttings were collected in Cameroon from cassava plants displaying cassava mosaic disease (CMD) symptoms. The nature of the viruses present was determined by using the PCR with primers specific for the coat protein (CP) genes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). All samples were infected by ACMV and eight of the 50 samples were infected by both ACMV and an EACMV-like virus. The complete nucleotide sequences of DNA-A and -B of representative ACMV and EACMV-like viruses were determined. The DNA-A component of the EACMV-like virus contained evidence of recombination in the AC2-AC3 region and DNA-B also contained evidence of recombination in BC1. However, both components retained gene arrangements typical of bipartite begomoviruses. When Nicotiana benthamiana plants were doubly inoculated with these Cameroon isolates of ACMV and EACMV (ACMV/CM, EACMV/CM) by using sap from cassava plants or infectious clones, the symptoms were more severe than for plants inoculated with either virus alone. Southern blot analysis of viral DNAs from infected plants showed that there were significantly higher levels of accumulation of both ACMV/CM components and, to a lesser extent, of EACMV/CM components in mixed-infected plants than in singly infected plants. These results strongly suggest the occurrence of a synergistic interaction between the two viruses.

Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content.

Chlorogenic acids (CGA) are involved in the bitterness of coffee due to their decomposition in phenolic compounds during roasting. CGA mainly include caffeoyl-quinic acids (CQA), dicaffeoyl-quinic acids (diCQA) and feruloyl-quinic acids (FQA), while CQA and diCQA constitute CGA sensu stricto (CGA s.s.). In the two cultivated species Coffea canephora and Coffea arabica, CGA s.s. represents 88% and 95% of total CGA, respectively. Among all enzymes involved in CGA biosynthesis, caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is not directly involved in the CGA s.s. pathway, but rather in an upstream branch leading to FQA through feruloyl-CoA. We describe how a partial cDNA corresponding to a CCoAOMT encoding gene was obtained and sequenced. Specific primers were designed and used for studying polymorphism and locating the corresponding gene on a genetic map obtained from an interspecific backcross between Coffea liberica var. Dewevrei and Coffea pseudozanguebariae. Offspring of this backcross were also evaluated for the chlorogenic acid content in their green beans. A 10% decrease was observed in backcross progenies that possess one C. pseudozanguebariae allele of the CCoAOMT gene. This suggests that CGA s.s. accumulation is dependent on the CCoAMT allele present and consequently on the activity of the encoded isoform, whereby CGA accumulation increases as the isoform activity decreases. Possible implications in coffee breeding are discussed.

Partial desiccation of mature embryo-derived calli, a simple treatment that dramatically enhances the regeneration ability of indica rice.

Regeneration of indica rice varieties remains a limiting factor for researchers undertaking rice Iransformation experiments. As reported for japonica rice and other crops, partial desiccation of indica rice calli dramatically promotes organogenesis and leads to high regeneration ability. We are now able to obtain 66.5%, 61.1% and 73.7% of calli that regenerate plants for the indica varieties TN1, IR72 and IR64 whereas in non desiccated controls only 30.0%, 15.5% and 18.7% of calli regenerated, respectively. Plants obtained were phenotypically normal and 50% were highly fertile. Partial desiccation is a reliable and simple method for improving indica rice regeneration. It also shortens the time of in vitro culture.

Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.

Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses.

Rice tungro disease is caused by an infection of two different viruses, rice tungro spherical virus (a (+) sense RNA virus) and rice tungro bacilliform virus (RTBV) with a genome of circular double-stranded DNA. The genome of an RTBV isolate from the Philippines was cloned, sequenced, and found to be 8000 bp in length. It contains four open reading frames (ORFs) on a single strand, with ORF 1 having an internal termination codon (TAA). The 5' and 3' ends of a polyadenylated viral RNA transcript, of genome length, were mapped by primer extension and cDNA sequence analysis, respectively. The transcript is terminally redundant by 265-268 nucleotides. Purified virus particles contain two major proteins with molecular masses of 37 and 33 kDa, although only the 37-kDa protein was detected in the infected rice tissues. The N-terminal amino acid sequence of the 33-kDa protein was determined and its coding region was identified on the RTBV genome. The identity of the coat protein gene was further confirmed by expressing a region of the genome in Escherichia coli, the products of which reacted with anti-RTBV antibody. The unusually long ORF 3 of RTBV is predicted to encode a polyprotein of 194.1 kDa that includes: the coat protein(s), viral proteinase, reverse transcriptase, and ribonuclease H. The sections of the polyprotein show varying degrees of similarity to the counterparts of Commelina yellow mottle virus (a member of the proposed badnavirus group) and caulimoviruses. The functions of the other three ORFs are unknown.