diversity in two
variants of Orobanche gracilis Sm. [var. gracilis
and var. deludens (Beck) A. Pujadas] (Orobanchaceae) from
different regions of
José Ignacio Cubero
Financial support: The work has been carried out at CIFA "Alameda del Obispo" (IFAFA-Área de Mejora y Biotecnología) and was supported by the INIA RTA04-067 project.
Keywords: AMOVA, molecular markers, Orobanche gracilis, parasitic plants, population genetics.
pattern of genetic variation among populations of two Orobanche
gracilis Sm. taxa (var. gracilis and var. deludens
(Beck) A. Pujadas) from Northern and
species are plants adapted to a parasitic mode of life. Most of the
species within Orobanche genus are found in the natural vegetation
(Pujadas-Salvà, 2002) and do not, or only occasionally,
occur on crop species. Nevertheless some of them have abandoned their
natural hosts and became very noxious parasitic weeds, causing major
crop losses. In this sense, O. crenata Forsk. causes huge damage
to legume crops (faba bean, lentil, pea and common vetch); O.
Although the economic importance has focused most of the research in this group mainly on physiological and applied issues, there are also many other aspects of both theoretical and practical interest that should be considered. In this sense, the study of natural O. populations can help us to understand gene pool dynamics, population size structure and geographical distribution, environmental adaptation and centres of origin. The parasitism has led to a simplification in the morphology of the genus (no leaves and only false roots) and therefore to a reduction in features used to distinguish species. Morphological traits are, therefore, of limited use to diversity studies due to its variation with environmental changes and estimation errors, and the reduced number of characteristic features available. In this context, the stability and the power of nucleic acid markers provides a clear advantage that has been exploited in different population studies of the genus, mainly considering species growing on crops of economical relevance such as O. cumana on sunflower (Gagne et al. 1998), O. crenata on legumes (Román et al. 2001; Román et al. 2002), or O. minor on clover (Westwood and Fagg, 2004).
In comparison with allozymes and microsatellites, RAPD markers present some limitations such as marker allele dominance and sometimes low reproducibility. Nevertheless, the major advantage of RAPD analysis out weight its disadvantage since it can potentially provide a much higher number of marker loci and higher levels of polymorphism than allozymes and costs much less being faster and easier to perform that microsatellite analysis because no prior DNA sequence information for the target species is required. Under controlled reaction conditions, reproducible and interpretable RAPD banding patterns can be obtained.
Molecular data on the interaction between parasitic plants and their native species which have been growing together for thousands of years are of great interest, not only to determine evolution patterns but also because these host species might have evolved ways of avoiding parasitism along evolution. It is not known how parasitic plants, which once occurred in the natural vegetation and apparently did not have a devastating effect on their host plant, escapes their "natural" environment and became harmful in crops. Some of these weedy species are still natives through most of their ranges.
gracilis Sm. grows on a wide range of wild Leguminosae scrubs
hosts such as Adenocarpus hispanicus, Anthyllis cytisoides
L., Astragalus granatensis Lam., Coronilla juncea L.,
Dorycnium pentaphyllum Scop., Erinacea anthyllis Link,
Genista cinerea (Vill.) DC., G. falcata Brot., G.
The aim of our present work is to study the genetic variation of these two varieties (var. gracilis and var. deludens) in order to (1) determine the genetic differences between them, (2) infer the inter and intra-population variability of both variants and (3) to determine the geographical influence on genetic diversity in distant populations of O. gracilis var. gracilis.
total of 166 specimens from 19 O. gracilis populations of two
O. gracilis taxa (var. gracilis and var. deludens)
were collected on different hosts over distinct regions of
RAPD analysis. Orobanche floral buds were used for DNA extraction using the method used by Román et al. (2001). For RAPD analysis, approximately 20 ng of genomic DNA was used as a template in a 25 µl volume per PCR reaction. Mixture composition and reaction conditions were as described by Román et al. (2001). Products were amplified in a Termocycler PE Applied Biosystems GeneAmp 9700.
A total of 15 primers of 10 bases length were analysed (OPA08, OPB03, OPB11, OPC18, OPD01, OPE08, OPF11, OPI19, OPJ01, OPN05, OPP12, OPQ18, OPR20, OPT07 and OPV09). Primers were purchased in commercially available kits from OPERON Technologies (Alameda, USA). Amplified products were electrophoresed on 1% agarose, 1% Nu-Sieve agarose, 1 x TBE gels, and visualised by ethidium bromide staining. Bands were scored manually using the Kodak Digital Science 1D Software program.
Statistical analysis. Amplified fragments were scored for the presence (1) or absence (0) of homologous bands to create a binary matrix of the different RAPD phenotypes. Shannon's information index as a measure of RAPD band diversity within populations was calculated as H0 = -Σ (pi log2 pi), where pi is the phenotypic frequency.
The matrix of inter-individual Dice's distance coefficients (Dice, 1945; Nei and Li, 1979) was subjected to a principal co-ordinate analysis (PCoA). From the distance matrix, new independent axial co-ordinates, which represent most of the variability of the original data, were calculated using NTSYS-pc ver. 2.1 (Rohlf, 2000). The individuals were then plotted as points in a two-dimensional continuous space defined by the first two co-ordinates.
The Dice's distance matrix was analysed by the analysis of molecular variance (AMOVA) approach using WINAMOVA 1.55 program. Total genetic variation was partitioned according to subspecies/regions and populations. The significance of φ-statistics was obtained non-parametrically by 1000 permutations. Homogeneity of intra-group molecular variances (homoscedasticity) was tested using the HOMOVA procedure (modified Barlett’s test), also carried out in WINAMOVA (Stewart and Excoffier, 1996). Modified Barlett’s statistics null distributions were obtained after 1000 permutations.
RAPD analysis proved to be an efficient method for obtaining information on the population genetic of O. gracilis. The 15 RAPD primers generated a total of 123 reliable fragments in the 19 analysed populations, after excluding bands that were monomorphic for the whole data set. Reliability was assessed by the maintenance of each polymorphic band in the total number of gels analysed. The number of bands per primer varied from 6 (OPI19 and OPJ01) to 15 (OPB03 and OPB15) with an average of 8.2 bands per primer. The size of the fragments ranged between 2083 and 275 bp. The proportion of polymorphic loci varied among populations from 30.89% to 67.48%. None of the populations considered showed unique bands. Two of the RAPD primers analysed in this study (OPB03 and OPV09) also amplified well with other Orobanche species such as O. crenata (Román et al. 2001) and O. foetida (Román et al. 2006) and could be considered in future broomrape studies on population biology.
diversity analysis within populations using
These results indicate a higher level of diversity in the populations from the North when compared to the South ones as well as a higher level of variation in the populations of the var. gracilis when compared to the var. deludens. Although the two groups of var. gracilis from the North and South showed a similar percentage of polymorphic loci (45.8% and 44.2%), the Shannon indexes remained different (0.27 and 0.24) since this value not only depends on the proportion of polymorphic loci but also on the distribution of allelic frequencies in the populations.
The estimated Dice distance coefficients varied from 0.0175 to 1 with an average value of 0.5697. The average distances among samples were plotted in a two-dimensional space using PCoA analysis (Figure 1). The general grouping clearly established the separation of samples according to the taxonomical variety and the geographical origin of each population. In this sense, the first PC1 that explained 21.24% of the total variation, clearly divided the three groups of individuals according to the botanical classification (var. gracilis from the N and S against var. deludens) (Table 3). In the case of PC2 explaining 20.19% of the total variation, this axial coordinate separated the populations by their geographical origin, clearly distinguishing populations from the North and South of Spain. The cumulative value of the two coordinates was 41.44%, being 7.57% and 6.82% the explained percentages of variation of the third and fourth ones.
The partition of variation in each group was studied with the analysis of the Dice's distance matrix by the AMOVA approach and a hierarchical analysis of phenotypic diversity using a two-way nested AMOVA was performed. In order to clarify the different hierarchical ranges, we distributed the populations in three different groups: O. gracilis var. gracilis from the North, O. gracilis var. gracilis from the South and O. gracilis var. deludens from the South. We first determined the general variation among the groups and populations and then analysed the partition of the total variation among the three groups considered. Moreover, the pairwise group partitions were also determined in order to verify the PCoA analysis.
The general AMOVA (Table 4) revealed that although most of the genetic diversity was attributable to differences among individuals within populations (46.52%), there was still a considerable level of variation among groups (27.23%) and populations within groups (26.26%) that showed significant associated φst values. The HOMOVA analysis also indicated that the molecular variances were heterogeneous among groups and populations within groups, suggesting clear differences among them.
The AMOVA analysis was also performed in each one of the three groups considered in order to determine the genetic structure of their populations (Table 5). In this sense, the analysis showed that in the populations from the North, the higher level of variation is found within population (80.09%) whereas only a 19.91% of the variation could be attributed to variation among populations. Both taxonomical varieties from the South (var. gracilis and var. deludens) presented a higher percentage of variation distributed among populations within the group (38.39% and 48.40% respectively) thus indicating a higher phenotypic structure. The higher level of variation shared among var. deludens populations, as well as the higher value of the associated φst statistic, shows a higher genetic differentiation among the populations of this taxonomical variant when compared to the other two groups. In all the cases considered, the values of the φst statistic and Barlett index were significant.
from the two AMOVA analysis described above, a third one determining
the proportion of variation between groups was performed (Figure
1). The most different groups were those that shared the higher
proportion of the total variance (29.10%) corresponding to var.
gracilis_South and var. deludens_South, where the percentage
of variation shared between the two taxonomical variants from the
North and the South was 27.95%. Finally, the most similar groups were
those with populations of O. gracilis var. gracilis
from two distant regions of
Although this study shows that both taxonomical varieties can be distinguished according to the molecular genotyping, the partition of variation between groups belonging to different variants is only slightly higher (29.10% and 27.95%) when compared to the variation between groups belonging to the same taxonomical variety (25.45%). In this sense, considering the similar proportion of variation explained by the two principal coordinates (21.24% and 20.19%), the separation of the three groups regarding the taxonomical variety or the geographic origin seems to be almost the same. The high proportion of variation within populations of O. gracilis var. gracilis in the North could be masking the differences between both taxonomical varieties.
results detected a lower level of intrapopulation diversity in both
varieties from the South (var. gracilis and var. deludens)
contrasting with the high intrapopulation variability found in var.
gracilis in the North of Spain with 19.91% of variation shared
among populations. In other O. species, the proportion of variability
reported among populations presented a wide range of variation: 47%
we do not have evidence of the mating system of O. gracilis,
contrasting results seem to be obtained for both taxonomical varieties.
In the case of var. gracilis from the North, the low level
of differentiation among populations (19.91%) is similar to that reported
for other allogamous species of the genera such as O. crenata
(Román et al. 2001; Román et al. 2002).
Nevertheless, the same species in the South as well as the taxonomical
var. deludens, present a relatively higher level of interpopulation
differentiation (38.39% and 48.40% respectively) more in agreement
with the pattern of genetic variation in a selfing species such as
fact that the populations belonging to the same taxonomical variety
showed lower levels of intrapopulation variability in the South when
compared to the North, could be suggesting a founder effect, considering
that in general populations have greatest genetic diversity in their
native regions, with colonizing subpopulations showing less within
population variation (Amsellen et al. 2000). High
genetic variability could represent an advantage for a species adapting
to a new environment, allowing the species to establish and survive,
whereas a reduced genetic variation lowers the adaptability of a population
increasing the risk of extinction in an event of changes in habitat
conditions. While gene flow among populations increases the variation
within and decreases the variation between populations, genetic drift
acts reciprocally. Therefore we may speculate that the samples of
O. gracilis var. gracilis from the South originated
from the Northern ones where higher levels of variability have been
found. After this event, differences among O. gracilis var.
gracilis populations from both distant regions of
As far as we are concerned this is the first report dealing with the pattern of variation in O. population attacking wild species. In this case, the distribution of variability is only related to its ability to disperse their pollen and /or seeds since no human acts are supposed to be involved on its distribution through the movement of commercial seeds from one place to another. The distribution of variation in this type of populations should indicate more ancient events that those provided by parasites growing on crops.
In the future, the analysis of new O. gracilis populations covering new geographical regions and hosts, together with the development of more robust makers to study genetic diversity in O. populations such as codominant microsatellites markers, will led to better understand the evolution of parasitic plants of wild flora still not adapted to agroecosystems.
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