Variability of interactions between barrel medic (Medicago truncatula) genotypes and Orobanche species

A number of broomrapes ( Orobanche spp.) are major threats for grain legumes in the Mediterranean area. Previous studies have shown very high levels of resistance to Orobanche crenata in Medicago truncatula with little variation among accessions hampering, therefore, its use for genetic analysis. In order to identify alternative systems for ulterior genetic and genomic analysis, we studied early stages of the interaction between M. truncatula accessions and a range of Orobanche species. We found significant differences in the induction of germination on Orobanche aegyptiaca , Orobanche foetida var. broteri , Orobanche minor , Orobanche nana and Orobanche ramosa seeds. Accessions also varied in the number of O. aegyptiaca , O. crenata , O. foetida var. broteri , O. nana and O. ramosa attachments supported. Applications of the synthetic germination stimulant GR24 increased the germination level of Orobanche cumana and O. minor . No attachments were observed for any of these species because of physical barriers typical of host resistance. On the contrary, increase of O. nana germination by GR24 led to an increase in number of attachments albeit the normal development of the nodules was stopped in a later stage. The genetic variation observed for induction of germination and subsequent attachment will be useful for isolating and characterising genes involved in early stages of Orobanche –host plant interaction and for the study of the biosynthetic pathways of production of germination stimulants.     

Molecular Studies on Orobanche

The optimization of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in Orobanche was investigated and the results were applied to analysis of O. aegyptiaca, O. oxyloba, O. ramosa and three O. crenata collections gathered from different locations in Egypt, Genotypes were compared using 20 random primers. The polymorphisms detected were only between the tested species, while identical banding patterns were generated in collections of the same species.
Genetic similarity among species was estimated on the basis of the percentage of common bands between species and a dendogram was constructed using the unweighted pair grouping method.
Two primers were chosen as specific PCR primers to differentiate between the studied Orobanche species. The results of this study show that RAPDs can be used as a method for clarifying relationships between species of Orobanche .     

Genetic diversity in Orobanche crenata populations from southern Spain

The pattern of genetic variation within and among natural populations of broomrape (Orobanche crenata Forsk.) from southern Spain was analysed by RAPD markers. Hierarchical analysis of phenotypic diversity using AMOVA was performed to analyse the partitioning of the variation among populations and among individuals. Although most of the genetic diversity was attributable to differences among individuals within a population (94.29%), significant φ_st values among populations suggested the existence of phenotypic differentiation. Moreover, corresponding HOMOVA analysis revealed that molecular variances were significantly heterogeneous among populations although no clear grouping pattern could be established. These results are to be expected considering the predominant outcrossing behaviour of O. crenata. Received: 10 January 2001 / Accepted: 12 February 2001     

Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination

Six sunflower sesquiterpene lactone models which share structural features of the lactone rings of strigol and its synthetic analogues, the GR family, with different conformational flexibilities were tested as Orobanche cumana germination stimulants. Among them, parthenolide and 3,5-dihydroxydehydrocostus-lactone significantly increased O. cumana germination, presenting higher activity than GR-24, used as a standard in the germination bioassay. The effect of these two compounds is species-specific, showing no germination stimulant activity on other Orobanche spp. tested (O. crenata, O. ramosa and O. aegyptiaca). Data presented are discussed in terms of a structure-activity relationship.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Use of random amplified polymorphic DNA (RAPD) markers in the study of the parasitic weed Orobanche

Despite the tremendous economic impact of broomrapes (Orobanche spp.) on agriculture in many countries little is known of the pattern of genetic variation within this group of parasitic weeds. The present paper describes the use of RAPD markers for the study of five Orobanche species in agricultural fields in Israel. Pronounced genetic differentiation was found between the species, and RAPD markers were raised for the identification of each of them. Southern-hybridization patterns of RAPD products of the various species were used to confirm the interpretation. The same markers were valid both for broomrapes collected in agricultural fields and for those collected in natural habitats. The validity of the markers found for O. cumana and O. crenata was confirmed on plants of the same species that were collected in Spain. Parsimony analysis of 86 RAPD characters produced a tree that clearly distinguishes between the five studied Orobanche species, separates the two Orobanche species belonging to sect. Trionychon from those belonging to sect. Osproleon, and supports the separation of O. cumana from O. cernua and of O. aegyptiaca from O. ramosa.     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

RAPD-marking of genomes of representative Hordeum species

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum. High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgare sensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between the Hordeum species, between the H. spontaneum populations, and between regional H. vulgare cultivars and a dendrogram was constructed.     

Fatty acids and tocochromanols in seeds of Orobanche

The evaluation of tocochromanols (tocopherols and tocotrienols) in 49 accessions from 21 Orobanche species revealed three well separated groups. The first one, characterized by high gamma-tocotrienol content, included all the accessions of sect. Orobanche. The second one, exhibiting high gamma-tocopherol content, comprised the accessions of O. arenaria Borkh. and O. purpurea Jacq. (sect. Trionychon Wallr.). All the other accessions of this section presented high delta-tocopherol content. Differences for tocochromanol derivatives within sect. Trionychon were paralleled by differences in the fatty acid profile, with the high delta-tocopherol class having also a higher oleic to linoleic acid ratio.     

RAPD Analysis of the Genome in Species of the Genus Hordeum

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum.High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgaresensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between theHordeumspecies, between the H. spontaneumpopulations, and between regional H. vulgarecultivars and a dendrogram was constructed.     

Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers

The knowledge of population structure and genetic diversity of wild relatives of rice is needed to investigate their evolutionary history and potential use in breeding programs. Very little is known about the wild rice species ( Oryza spp.), particularly those that are native to South America. A study using isozyme and RAPD markers was conducted to estimate the level of genetic diversity of four South American wild rice populations ( Oryza glumaepatula ) recently collected in the Amazon forest and western Brazil rivers. F -statistics and genetic diversity parameters calculated from isozyme and RAPD markers indicated high values for inbreeding coefficients and differentiation among the four populations. In agreement with this, a pattern of greater variation between than within populations was observed with both types of markers. These findings were corroborated by an AMOVA analysis, which indicated that a large portion of the total genetic variation was attributed to regional divergence. The partition of the AMOVA analysis among populations showed that most of the genetic diversity was due to differences among populations. This distribution pattern of genetic variation of O. glumaepatula populations is in agreement with the expectation for an autogamous species and provides important baseline data for conservation and collection strategies for this species.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

Potential role of winter rape weeds in the extension of broomrape in Poitou-Charentes

In the Poitou-Charentes district, among the 82 species of winter rape weeds identified, 22 displayed a strong affinity for this crop (Brassica napus L.). In fields, 50% of these weeds were parasitized by Orobanche ramosa, playing the role of host plants. Greenhouse co-cultures (weed/Orobanche ramosa) showed that weeds non-parasitized in fields could be attacked by broomrape, developing a more or less complete cycle. In vitro co-cultures (weed/Orobanche ramosa) revealed that root exudates of non-parasitized weeds, in fields or in greenhouse co-cultures, could induce Orobanche ramosa seed germination, but not attachment. These weeds could play the role of false hosts.     

Isolated populations or isolated taxa? A case study in narrowly-distributed snapdragons (Antirrhinum sect. Sempervirentia) using RAPD markers

RAPD fingerprinting was used to study species boundaries in narrowly distributed endemic species in Antirrhinum section Sempervirentia. Based on RAPD data, similarity values within species were relatively high but pair-wise similarity values among species were low. Partitioning of the overall RAPD variation using AMOVA showed that most of the variation was found among species (58.06%), whereas the remaining phenotypic diversity was distributed among populations (25.18%) and among individuals within populations (16.76%). Comparison of the matrices of geographical distances and phenetic distances (1-Dice index) among populations using the Mantel test showed a moderate, but statistically significant correlation (r=0.588, P < 0.01), suggesting that isolation by distance is responsible for the distribution of genetic variation among Antirrhinum populations. Phenetic relationships among Antirrhinum samples based on a Dice similarity matrix showed a clear taxonomic pattern, confirming the grouping of individuals within their own populations and the clustering of populations within species. Individuals of A. charidemi, A. valentinum and A. subbaeticum, from subsection Valentina, made up a discrete group, whereas the samples belonging to subsection Sempervirentia (A. petegasii, A. sempervirens, A. microphyllum, A. pulverulentum) clustered together. RAPD data are entirely congruent with the subsection classification scheme proposed by Fernández Casas (1997) in section Sempervirentia. However, A. subbaeticum, treated as a synonym of A. valentinum by Fernández-Casas (1997), showed an unique RAPD profile characterized by the highest number of fixed species-specific markers found in section Sempervirentia. Thus, although A. valentinum appeared the most closely related species to A. subbaeticum, molecular data suggested that this species merits taxonomic distinction.     

Variability of Orobanche ramosa populations in France as revealed by cross infestations and molecular markers

The dramatic spread of root holoparasite Orobanche ramosa L. (branched broomrape) has been observed in the last few years in France in fields of Brassica napus L., Nicotiana tabacum L., and Cannabis sativa L. In the face of this occurrence and considering its tremendous capacity to adapt to its host, the question about variability of O. ramosa populations arises. The virulence of broomrape populations was investigated by cross infestations performed under greenhouse conditions using the three host species mentioned above and broomrape seeds collected from Orobanche plants parasitizing them. After 45 days of co-culture, the intensity and kinetics of infestation were determined. In all cases, parasites developed but differences were observed among associations between the host species and the broomrape population. Differences in virulence of O. ramosa populations and greater affinity between a host species and “its own” broomrape population were demonstrated by statistical analyses (F-test, CFAs). These results led to the concept of host specificity and suggestions about the existence of three different pathovars in O. ramosa. Analysis of genetic variability of the three O. ramosa populations by PCR of RAPD markers confirmed that they may be considered as three distinct pathovars.     

Host differentiation in Orobanche foetida Poir

Orobanche foetida Poir. is a parasitic plant widely distributed in the Western Mediterranean area. It typically parasitizes wild plants but has recently been described as an agricultural problem in legume crops in Tunisia. The pattern of genetic variation within and among O. foetida populations growing on chickpea and faba bean was analyzed by RAPD markers. The UPGMA cluster analysis based on Dice distance matrix showed a clear differentiation among O. foetida samples collected on chickpea and those on faba bean, suggesting a host-differentiation process. Although an AMOVA analysis revealed substantial internal variation among individuals within O. foetida populations (69.8%), there was a significant divergence between parasites on the two hosts considered (30.2%). Moreover, germination of O. foetida seeds collected on chickpea and faba bean in the presence of both host roots was studied. Germination percentages of O. foetida seeds varied depending on the host used both for collecting the seeds and evaluating the trait. According to these results, possible explanations for the origin of this new weedy parasite and the host differentiation process are discussed.     

An improved axenic system for studying pre-infection development of the parasitic plant Orobanche ramosa

BACKGROUND AND AIMS: Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control early stages of host infection in Orobanche are poorly understood, partly due to the lack of experimentally tractable in vitro systems that allow the efficient application of molecular tools. Here an improved axenic system for the analysis of pre-infection stages in O. ramosa in the absence of the host plant is described. METHODS: An optimized protocol for seed disinfection, based on formaldehyde, was developed. Orobanche ramosa seeds were conditioned in Petri dishes with filter paper, stimulated by addition of the synthetic strigol analogue GR24, and the percentage of germination as well as attachment-organ formation was determined. KEY RESULTS: Treatment of O. ramosa seeds with tobacco-root exudate or with GR24 resulted in highly reproducible germination rates around 70 %. A conditioning period of 8 d was both necessary and sufficient to allow optimal germination in response to GR24. Conditioned seeds that were dehydrated for several months remained fully responsive to GR24 without the need of a new conditioning period. Treatments as short as 5 min with GR24 were sufficient to fully and irreversibly induce the seed germination response. Approximately half of the germinated seeds initiated attachment-organ development. Similar rates of attachment organ induction were also detected in the rare cases of seeds that had germinated spontaneously on water. CONCLUSIONS: The results suggest that the conditioning period produces persistent changes in the seeds required for responsiveness to external stimulants. The rapid action of GR24 suggests that it may act via a receptor-mediated signalling mechanism. While germination in O. ramosa is induced by exogenous stimuli, attachment organ differentiation appears to be triggered by unknown endogenous signals. The new in vitro culture system will have useful applications for the molecular analysis of early stages of parasitic development in Orobanche.     

Implications of ITS sequences and RAPD markers for the taxonomy and biogeography of the Oxytropis campestris and O. arctica (Fabaceae) complexes in Alaska

Taxonomic consensus is lacking on the Oxytropis arctica and O. campestris species complexes, two polyploid complexes found in the interior and arctic areas of Alaska. One classification has emphasized flower size, whereas flower color is considered a key diagnostic character in another classification. Our analyses of internal transcribed spacer (ITS) sequences and random amplified polymorphic DNA (RAPD) markers provided no support for either classification system. The trees generated from ITS sequences and the phenogram derived from RAPD markers suggest that most recognized taxa in the two complexes are probably polyphyletic, including O. arctica var. barnebyana, which is listed as threatened in Alaska. The only consistent pattern detected by both types of molecular markers was a geographic split dividing the northeastern arctic populations from most other populations (48.60-55.03% in AMOVA analyses). This genetic subdivision probably reflects a Pleistocene barrier formed by the northern coastal ice shield. Our molecular data, in conjunction with the previously reported variation of ploidy levels in these groups, suggest a scenario of recent and multiple origins of polyploidy. It is possible that most Alaskan populations of these two complexes are best referred to as a single taxonomic species despite morphological differentiation within the complexes.     

Morphological and RAPD analysis of hybridization between Quercus affinis and Q. laurina (fagaceae), two Mexican red oaks

Quercus affinis and Q. laurina are two closely related Mexican red oaks with partially overlapping distributions. Within the area of overlap, there are localities where morphological intergradation occurs. A previous hypothesis explained this pattern as a result of secondary contact between the two species, followed by hybridization and introgression. This possibility was analyzed here by examining foliar and genetic variation in 16 localities situated along a macrogeographic gradient, which included morphologically representative populations of both species and populations from within the area of overlap. Maximum-likelihood hybrid index scores calculated from nine semi-diagnostic RAPD markers indicated a shift in the genetic composition of populations from one species to the other along the macrogeographic gradient, with genetically intermediate populations situated in the area of overlap. Foliar variation followed a partially congruent pattern, but Q. laurina-like morphology predominated in some of the genetically intermediate populations. There were several instances of correlated frequency changeovers of single RAPD markers and morphological characters along the macrogeographic gradient and a few cases of markedly parallel patterns between markers. The results were interpreted as consistent with a hypothesis of secondary contact between the two oak species that has resulted in some differential introgression among markers.     

Correlated evolution of life history and host range in the nonphotosynthetic parasitic flowering plants Orobanche and Phelipanche (Orobanchaceae)

Most members of the nonphotosynthetic parasitic genera Orobanche and Phelipanche (Orobanchaceae) have narrow host ranges, and, as they grow on perennial hosts, are (at least potentially) perennial themselves. A few species, however, have wide host ranges and grow on annual hosts, and are thus (at least facultatively) annuals themselves. Among the latter are the weedy species, which include economically important pest taxa such as Orobanche crenata or Phelipanche aegyptiaca. Using a phylogenetically based maximum likelihood approach, which takes phylogenetic and branch length uncertainty into account, we can show that the life trait host range and life history evolve in a correlated fashion. This supports the hypothesis that parasite specialization is associated with predictable resources (i.e. long-lived hosts) and generalism with unpredictable ones (i.e. short-lived hosts), a pattern often found in animal parasites. The mechanisms and temporal sequence of the life trait changes and their interrelations remain speculative.