Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms.

The domestication of the Eurasian grape (Vitis vinifera ssp. sativa) from its wild ancestor (Vitis vinifera ssp. sylvestris) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations.

Variability and genetic structure of the population of watermelon mosaic virus infecting melon in Spain.

The genetic structure of the population of Watermelon mosaic virus (WMV) in Spain was analysed by the biological and molecular characterisation of isolates sampled from its main host plant, melon. The population was a highly homogeneous one, built of a single pathotype, and comprising isolates closely related genetically. There was indication of temporal replacement of genotypes, but not of spatial structure of the population. Analyses of nucleotide sequences in three genomic regions, that is, in the cistrons for the P1, cylindrical inclusion (CI) and capsid (CP) proteins, showed lower similar values of nucleotide diversity for the P1 than for the CI or CP cistrons. The CI protein and the CP were under tighter evolutionary constraints than the P1 protein. Also, for the CI and CP cistrons, but not for the P1 cistron, two groups of sequences, defining two genetic strains, were apparent. Thus, different genomic regions of WMV show different evolutionary dynamics. Interestingly, for the CI and CP cistrons, sequences were clustered into two regions of the sequence space, defining the two strains above, and no intermediary sequences were identified. Recombinant isolates were found, accounting for at least 7% of the population. These recombinants presented two interesting features: (i) crossover points were detected between the analysed regions in the CI and CP cistrons, but not between those in the P1 and CI cistrons, (ii) crossover points were not observed within the analysed coding regions for the P1, CI or CP proteins. This indicates strong selection against isolates with recombinant proteins, even when originated from closely related strains. Hence, data indicate that genotypes of WMV, generated by mutation or recombination, outside of acceptable, discrete, regions in the evolutionary space, are eliminated from the virus population by negative selection.

Variability and genetic structure of plant virus populations.

Populations of plant viruses, like all other living beings, are genetically heterogeneous, a property long recognized in plant virology. Only recently have the processes resulting in genetic variation and diversity in virus populations and genetic structure been analyzed quantitatively. The subject of this review is the analysis of genetic variation, its quantification in plant virus populations, and what factors and processes determine the genetic structure of these populations and its temporal change. The high potential for genetic variation in plant viruses, through either mutation or genetic exchange by recombination or reassortment of genomic segments, need not necessarily result in high diversity of virus populations. Selection by factors such as the interaction of the virus with host plants and vectors and random genetic drift may in fact reduce genetic diversity in populations. There is evidence that negative selection results in virus-encoded proteins being not more variable than those of their hosts and vectors. Evidence suggests that small population diversity, and genetic stability, is the rule. Populations of plant viruses often consist of a few genetic variants and many infrequent variants. Their distribution may provide evidence of a population that is undifferentiated, differentiated by factors such as location, host plant, or time, or that fluctuates randomly in composition, depending on the virus.

Molecular epidemiology of Cucumber mosaic virus and its satellite RNA.

Molecular analysis of viral isolates can yield information that facilitates an understanding of virus epidemiology and has been termed molecular epidemiology. This approach has only recently been applied to plant viruses. Results on the molecular epidemiology of Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) in Spain, where CMV is endemic in vegetable crops are presented here. To characterise the genetic structure of CMV populations, c. 300 isolates, representing 17 outbreaks (i.e. sub-populations) in different crops, regions and years, were compared. Genetic analyses of CMV isolates were done by ribonuclease protection assay of cRNA probes representing RNA1, RNA2 and the two open reading frames in RNA3. All isolates belonged to one of three genetic types: Sub-group II and two types of Sub-group I. The genetic structure of the 17 sub-populations varied randomly, without correlation with location, year, or host plant species. Thus, CMV in Spain shows a metapopulation structure with local extinction and random recolonisation from local or distant virus reservoirs. The frequency of mixed infections and of new genetic types generated by reassortment of genomic segments or by recombination was also estimated. Results indicate that heterologous genetic combinations are not favoured. About 30% of CMV isolates were supporting a satRNA. The frequency of CMV isolates with a satRNA differed for each sub-population, being c. 1 in eastern Spain in 1990 and decreasing to c. 0 in distant regions and in subsequent years. Molecular analyses of CMV-satRNA isolates show high genetic diversity, due both to the accumulation of point mutations and to recombination. The CMV-satRNA population is a single, unstructured one. Thus, the CMV-satRNA population has a genetic structure and dynamics different from those of its helper virus. This indicates that CMV-satRNA has spread epidemically on the extant virus population from an original reservoir in eastern Spain. The relevance of these results for the control of CMV infections is discussed.

More than 80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana.

Transcription factors belonging to the R2R3-MYB family contain the related helix-turn-helix repeats R2 and R3. The authors isolated partial cDNA and/or genomic clones of 78 R2R3-MYB genes from Arabidopsis thaliana and found accessions corresponding to 31 Arabidopsis genes of this class in databanks, seven of which were not represented in the authors' collection. Therefore, there are at least 85, and probably more than 100, R2R3-MYB genes present in the Arabidopsis thaliana genome, representing the largest regulatory gene family currently known in plants. In contrast, no more than three R2R3-MYB genes have been reported in any organism from other phyla. DNA-binding studies showed that there are differences but also frequent overlaps in binding specificity among plant R2R3-MYB proteins, in line with the distinct but often related functions that are beginning to be recognized for these proteins. This large-sized gene family may contribute to the regulatory flexibility underlying the developmental and metabolic plasticity displayed by plants.

Satellite RNA of cucumber mosaic cucumovirus spreads epidemically in natural populations of its helper virus.

ABSTRACT Three hundred thirty-eight isolates of cucumber mosaic cucumovirus (CMV), sampled from natural populations in six areas of Spain between 1989 and 1996, were screened for the presence of satellite RNA (satRNA). The frequency of CMV isolates with satRNA approached 1.00 in Valencia (east Spain) between 1990 and 1994 where a tomato necrosis epidemic induced by CMV+satRNA had started in 1986 and was smaller north and west of this area in 1992 and 1993. After 1994, satRNA almost disappeared from all CMV populations. Genetic typing of satRNA variantswas done by ribonuclease protection assay, and from these data, genetic distances were estimated for any pair of satRNA variants. CMV-satRNA populations were highly diverse, containing 0.07865 nucleotide substitutions per site on average. Data also showed that the whole compared set of 100 satRNA variants form a single population that is not structured according to place, year, host plant, or strain of helper virus (HV). This is in sharp contrast with the metapopulation structure of the Spanish CMV population. Thus, the genetic structure and dynamics of populations of CMV and its satRNA are not coupled. This shows that CMV-satRNA spreads epidemically, as a hyperparasite, in the population of its HV. This conclusion is relevant to the use of CMV-satRNA as a biocontrol agent of CMV.

A mutation in tomato aspermy cucumovirus that abolishes cell-to-cell movement is maintained to high levels in the viral RNA population by complementation.

The nucleotide substitution C-->A at nucleotide 100 of tomato aspermy cucumovirus (TAV) strain V (V-TAV) RNA segment 3 (RNA3) introduces an ocher stop at the fourth codon of the movement protein open reading frame. Experiments with RNA transcripts from full-length clones showed that this mutation abolished cell-to-cell movement and, thus, infectivity in planta. Heterogeneity analyses on stock V-TAV virion RNA showed that an A at position 100 was present in the molecular population of RNA3 at a frequency of 0.76 and that a C at this position was present at a frequency of 0.24. This result indicates that a fraction of RNA3 molecules complements cell-to-cell movement of movement-defective molecules. It was shown that the mutation C-->A conferred enhanced RNA replication of the defective mutant in tobacco protoplasts. The effect of the mutation on replication was dependent on sequence context, since the same mutation did not affect the replication efficiency in the related TAV strain 1 RNA3. Competition experiments in tobacco protoplasts were done to estimate the fitness during a cell invasion cycle of the movement-defective mutant relative to the wild type (wt). From these data, a lower limit to the degree of complementation of movement-defective molecules by movement-competent ones could be estimated as 0.13. This estimate shows that complementation may play an important role in the determination of genetic structure in RNA genome populations. A further effect of the enhanced replication of the movement-defective mutant was the efficient competition with the wt for the initiation of infection foci in planta.

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.

Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus.

Two hundred seventeen field isolates of cucumber mosaic cucumovirus (CMV), sampled from 11 natural populations, were typed by RNase protection assay (RPA) using probes from the genomic RNAs of strains in subgroup I and in subgroup II of CMV strains. Most (85%) of the analyzed isolates belonged to subgroup I. For these subgroup I isolates, only two clearly different RPA patterns, A and B, were found for each of four probes representing RNA1, RNA2, and each of the two open reading frames in RNA3. On the basis of these RPA patterns for each probe, different haplotypes were defined. The frequency composition for these haplotypes differed for the various analyzed populations, with no correlation with place or year of sampling. This genetic structure corresponds to a metapopulation with local extinctions and recolonizations. Most subgroup I isolates (73%) belonged to haplotypes with RPA pattern A (type 1) or B (type 2) for all four probes. A significant fraction of subgroup I isolates (16%) gave evidence of mixed infections with these two main types, from which genetic exchange could occur. Genetic exchange by segment reassortment was seen to occur: the fraction of reassortant isolates was 4%, reassortment did not occur at random, and reassortants did not become established in the population. Thus, there is evidence of selection against reassortment between types 1 and 2 of subgroup I isolates. Aphid transmission experiments with plants doubly infected with type 1 and type 2 isolates gave further evidence that reassortment is selected against in CMV. Genetic exchange by recombination was detected for RNA3, for which two RPA probes were used. Recombinant isolates amounted to 7% and also did not become established in CMV populations. Sequence analyses of regions of RNA1, RNA2, and RNA3 showed that there are strong constraints to maintain the encoded sequence and also gave evidence that these constraints may have been different during divergence of types 1 and 2 and, later on, during diversification of these two types. Constraints to the evolution of encoded proteins may be related to selection against genetic exchange. Our data, thus, do not favor current hypotheses that explain the evolution of multipartite viral genomes to promote genetic exchange.

Contribution of mutation and RNA recombination to the evolution of a plant pathogenic RNA.

The nucleotide sequence of 17 variants of the satellite RNA of cucumber mosaic virus (CMV-satRNA) isolated from field-infected tomato plants in the springs of 1989, 1990, and 1991 was determined. The sequence of each of the 17 satRNAs was unique and was between 334 and 340 nucleotides in length; 57 positions were polymorphic. There was much genetic divergence, ranging from 0.006 to 0.141 nucleotide substitutions per site for pairwise comparisons, and averaging 0.074 for any pair. When the polymorphic positions were analyzed relative to a secondary structure model proposed for CMV-satRNAs, it was found that there were significantly different numbers of changes in base-paired and non-base-paired positions, and that mutations that did not disrupt base pairing were preferred at the putatively paired sites. This supports the concept that the need to maintain a functional structure may limit genetic divergence of CMV-satRNA. Phylogenetic analyses showed that the 17 CMV-satRNA variants clustered into two subgroups, I and II, and evolutionary lines proceeding by the sequential accumulation of mutations were apparent. Three satRNA variants were outliers for these two phylogenetic groups. They were shown to be recombinants of subgroup I and II satRNAs by calculating phylogenies for different molecular regions and by using Sawyer's test for gene conversion. At least two recombination events were required to produce these three recombinant satRNAs. Thus, recombinants were found to be frequent ( approximately 17%) in natural populations of CMV-satRNA, and recombination may make an important contribution to the generation of new variants. To our knowledge this is the first report of data allowing the frequency of recombinant isolates in natural populations of an RNA replicon to be estimated.

Genetic diversity in tobacco mild green mosaic tobamovirus infecting the wild plant Nicotiana glauca.

The variability and genetic structure of tobacco mild green mosaic tobamovirus (TMGMV) was analyzed in a collection of isolates from Nicotiana glauca plants from Australia, California, Spain, and the east Mediterranean Basin. Ribonuclease protection was assayed on 53 isolates with six probes representing 85% of the TMGMV genome. Results showed that conserved domains in the TMGMV genome were different for each geographic population. The nucleotide sequence of two regions of the TMGMV genome was determined for 33 isolates. Nucleotide diversity values were smaller than those reported for other RNA genomes. For each population, genetic diversity was not related to the size of the area from which the isolates were collected, and was particularly small for Australia and Spain. Diversity values indicated population differentiation, which was largest between the less diverse Spanish and Australian populations. Nucleotide diversity values for nonsynonymous vs synonymous positions indicate moderate negative selection, except for the Spanish population for which it gave evidence of positive selection. On the whole, our data indicate that the low genetic diversity for this plant virus may be due to different factors such as positive and negative selection or the recent colonization of new areas or host plants. The intensity of these factors appears to be different for the various TMGMV geographical populations.

Rye flour allergens associated with baker's asthma. Correlation between in vivo and in vitro activities and comparison with their wheat and barley homologues.

BACKGROUND: A number of wheat and barley flour proteins that belong to the cereal alpha-amylase/trypsin inhibitor family have been identified as major allergens associated with baker's asthma. However, the allergenic role of this protein family had not been investigated in rye. OBJECTIVE: To study the allergenicity of five purified proteins from rye flour which belong to the same inhibitor family, as well as to compare their properties with those of their wheat and barley homologues. METHODS: In vivo skin-prick tests were carried out in 21 patients with radioallergosorbent test (RAST) 2-3 to rye and allergic sensitization mainly to this cereal flour. In addition, sera from all these patients were used to assay the IgE binding capacity of dot blotted purified proteins. RESULTS: Three of the rye proteins tested, namely Sec c 1, RDAI-1 and RDAI-3, provoked positive skin-prick tests in more than 50% of patients, although their in vitro reactivity was lower. Different reactivities were found for the rye components compared with their wheat and barley homologues. Statistical analyses showed a significant correlation between the results of in vivo and in vitro tests for seven out of the nine purified proteins considered in this study. CONCLUSION: Members of the rye alpha-amylase inhibitor family are main allergens involved in allergic reactions to cereal flours. However, different allergenic behaviours were found between homologous allergens from rye, barley, and wheat.

Inhibitory activities against heterologous α-amylases and in vitro allergenic reactivity of Einkorn wheats.

Salt extracts from seeds of 36 lines of Einkorn wheats were analyzed for their inhibitory activity towards two insect (Tenebrio molitor, Coleoptera, and Ephestia kuehniella, Lepidoptera) and one mammalian (human salivary) α-amylases. Whereas all ten T. monococcum accessions tested were active towards the lepidopteran enzyme, they had no effect on the coleopteran or the mammalian ones. More variability was found among the 21 lines of T. boeticum analyzed, although none of them inhibited human α-amylase. The five accessions of T. urartu showed even greater diversity. Among all Einkorn accessions tested, only two urartu lines affected the three α-amylases. These lines displayed inhibition patterns similar to those of T. aestivum and T. turgidum cultivars. Since several breadwheat α-amylase inhibitors are major allergens associated with baker's asthma, we also studied the in vitro allergenic activity of salt extracts from the Einkorn wheats under study. No significant differences in IgE-binding were found between these accessions and theT. aestivum or T. turgidum cultivars. Furthermore, putative allergens with molecular sizes in the range of 20-60 kDa were detected in these Einkorn wheats.

Experimental evaluation of the ribonuclease protection assay method for the assessment of genetic heterogeneity in populations of RNA viruses.

The ribonuclease (RNase) protection assay (RPA) was evaluated as a method to estimate genetic distances among sequence variants of RNA viruses. The patterns of fragments generated, under different RPA conditions, by three sets of RNA sequence variants of known nucleotide sequence, were analyzed. Both the effectiveness of cleavage (i.e. the probability of cleavage in a certain heteroduplex) and its degree (i.e. in all the molecules in the assay or in a part of them) varied largely according to the nature of the mismatch. Probability and degree of cleavage were also dependent on distant sequence context effects. No correlation could be established between context and cleavage, so that the pattern of fragments in RPA cannot be unequivocally predicted from sequence information. Accordingly, nucleotide sequence differences between two sequence variants cannot be directly derived from RPA data. For all three sequence sets linear relationships were found between the number of non-shared fragments in the RPAs of two variants and their nucleotide sequence differences. Nevertheless, both linearity and the linear regression parameters varied largely according to the sequence set and according to RPA conditions, in a non-predictable way. Thus, under experimental conditions, RPA may not be as appropriate a method to estimate genetic distances between RNA sequences as simulation under an ideal model suggested. Possible ways to diminish the gap between the ideal model and the experimental procedure are proposed.

On recombination among In(2L)t, alpha-Gpdh and Adh in Drosophila melanogaster.

The occurrence and patterns of linkage disequilibrium between an inversion and allozymic loci within it or nearby have been used in attempts to discriminate among different hypotheses for the maintenance of variability. The interpretation of the data on the best-documented case, that of the nonrandom association between In(2L)t and alpha-Gpdh or Adh in the second chromosome of Drosophila melanogaster, has been done on the basis that recombination between alpha-Gpdh and Adh is almost entirely due to the recombination between In(2L)t and the locus within it (alpha-Gpdh), the recombination between the inversion and the nearby locus (Adh) being negligible. In this paper, we show that the pattern of recombination is just the opposite.

The nucleotide sequence of a HMW glutenin subunit gene located on chromosome 1A of wheat (Triticum aestivum L.).

A cloned 8.2 kb EcoRI fragment has been isolated from a genomic library of DNA derived from Triticum aestivum L. cv. Cheyenne. This fragment contains sequences related to the high molecular weight (HMW) subunits of glutenin, proteins considered to be important in determining the elastic properties of gluten. The cloned HMW subunit gene appears to be derived from chromosome 1A. The nucleotide sequence of this gene has provided new information on the structure and evolution of the HMW subunits. However, hybrid-selection translation experiments suggest that this gene is silent.

A two loci viability matrix with implications in current population genetics models.

Some equilibrium properties of a two loci deterministic system subject to a postulated viability matrix are given. Two conclusions are reached: i) linkage can not stabilize additive equally contributing two-loci systems under optimizing selection. ii) Hybrid protein superiority can not be invoked for the maintenance of polymorphisms shared by duplicated structural loci.