morphic DNA fragments by the polymerase chain reaction (PCR) using a single oligonucleotide primer
of arbitrary sequence [79]. RAPD markers have already been used to differentiate between important
species of the genus Orobanchein Israel [31,32,80].
B. Establishing of Orobanche Ecotypes
So far, all molecular studies of Orobancheparasitic plants have been performed with plant material from
one generation and usually so-called origins have been used, presumably representing a mixture of para-
sitic plant seeds as no inbred lines have been prepared for the parasite. Because of their mode of life, par-
asitic plants can reproduce by self-pollination [1,18,19], which makes it easy to produce plants with a rel-
atively high degree of homozygosity and homogeneity. Such ecotypes are appropriate to trace inheritance
of molecular markers and give more precise answers on species discrimination questions. They can also
be used for other types of genetic research.
We produced ecotypes for three species from section Trionycon Warll.—O. ramosa, O. aegyptiaca,
andO. oxyloba—and studied representatives of the last two generations by RAPD analysis for the amount
of genetic variability among the ecotypes and between the generations. We examined representatives of
section Trionycon of the genus Orobanchebecause it is considered the “ancestral stock” on the basis of
its chromosome number, 2n24 [54].
ThreeO. ramosagenotypes with different geographic origins (Spain, Bulgaria, and North America)
were chosen for the study as they represent highly isolated populations of the species. In this way there
was a possibility to trace the partition of variation between populations as a function of the geographical
distance between the regions of collection.
AnO. aegyptiacagenotype was used as a closely related species in order to obtain more information
about the disputable species complex O. ramosa/O. aegyptiaca.
O. oxylobais a species not yet reported to damage the crop yield, so it is most interesting to compare
it with the other two in order to see the degree of relatedness between the three representatives of the same
section of the genus Orobanche. Besides, there are no data about its chromosome number and an as-
sumption has been made that a comparison could throw light on the evolutionary pathways within the
genus [54].
Four generations of plants have been obtained, thus showing that all three species can reproduce by
self-pollination. As the lives most suitable for RAPD mapping are inbred lines derived from repeated self-
ing of individual plants of an F 2 population [81], the study was performed on those and also on plants
from F 3. Further selection was ceased to prevent inbreeding depression.
C. Reproducible Different DNA Fingerprints for All Ecotypes
The pattern of DNA polymorphisms (Figures 1 and 2) and the genetic distances that we observed (Table
1, Figure 3) demonstrate that RAPD analysis of the five Orobancheecotypes studied provides possibili-
ties to distinguish clearly between them, as single primers and primer combinations generated repro-
ducible different DNA fingerprints. These primers were selected because they had been used for similar
purposes in Israel [80] and Spain [82]. Thus, it is possible to claim that a set of primers is already estab-
lished that can be successfully used in different laboratories for identification of Orobanchespecies of
economic importance.
The results of our study indicate that the genetic distance (GD) between the O. ramosagenotype
cluster and the O. aegyptiaca/O. oxylobacluster is big enough (GD 0.72) to consider O. ramosaand
O. aegyptiacaunambiguously different species. This corresponds to the findings about populations of the
same species in Israel [80] and Egypt [31].
There are significant differences between the three O. ramosagenotypes. The most closely related
proved to be the populations from America and Bulgaria (GD 0.15). There is evidence that the Amer-
ican and European genera of Orobanchaceae do not share a common Orobanchaceae ancestor and thereby
have evolved at slightly different rates [83]. The reason for the closer link between the two populations
exhibited in our study could probably be the relatively recent introduction of this particular American
genotype in the New World habitat.
Spain is the westernmost site of Orobanchedistribution in the Mediterranean region. The reason for
the pronounced genetic distance of this genotype from the other two O. ramosagenotypes may be that the
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