Next Article in Journal
Path Coefficient and Principal Component Analyses for Biomass Allocation, Drought Tolerance and Carbon Sequestration Potential in Wheat
Previous Article in Journal
Different Concentrations of Potassium Silicate in Nutrient Solution Affects Selected Growth Characteristics and Mineral Composition of Barley (Hordeum vulgare L.)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 11
Issue 11
10.3390/plants11111406
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Molecular Characterization of a New Ecotype of Holoparasitic Plant Orobanche L. on Host Weed Xanthium spinosum L.

by
Ali Reza Yousefi
1,*,
Asadollah Ahmadikhah
2,
Reza Fotovat
1,
Leila Rohani
1,
Foad Soheily
1,
Daniela Letizia Uberti
3 and
Andrea Mastinu
3,*
1
Department of Plant Production & Genetics, University of Zanjan, Zanjan 45371, Iran
2
Department of Plant Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran 31587, Iran
3
Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy
*
Authors to whom correspondence should be addressed.
Plants 2022, 11(11), 1406; https://doi.org/10.3390/plants11111406
Submission received: 7 April 2022 / Revised: 15 May 2022 / Accepted: 24 May 2022 / Published: 25 May 2022
(This article belongs to the Section Plant Systematics, Taxonomy, Nomenclature and Classification)
Browse Figures
Review Reports Versions Notes

Abstract

:
A species of Orobanche was observed on spiny cocklebur (Xanthium spinosum) for the first time in Iran and tentatively was named IR-Iso.This study was conducted to make a phylogenetic analysis of the Orobanche using 5.8S rRNA region sequences, and also to better understand its sequence pattern. The full-length ITS1-ITS2 region of the new Orobanche isolate was PCR-amplified from the holoparasitic plant parasitizing X. spinosum. Sequences of the amplicons from the isolate were 100% identical but differed by 5.6–6.7% from most homologous GenBank accessions to 37.9% divergence from distant species. The analysis of the molecular variance showed that variation between-population (61.9%, SE = 0.04) was larger than within-population. Neighbor-joining analysis placed the Iranian isolate in the same clade as most of the Orobanche and Phelipanche species. The isolate was more closely related to Orobanche aegyptiaca (from China), and this was confirmed by using a structure analysis. However, complementary analyses showed that the Iranian isolate has a unique nucleotide substitution pattern, and hence it was considered as an ecotype of O. aegyptiaca (ecotype Alborzica). In this paper we report on the association between this new ecotype of Orobanche and X. spinosum.

1. Introduction

The Orobanchaceae family is the largest family of parasitic flowering plants, including nearly 2060 holo- or hemiparasitic species in 90–99 different genera [1,2,3,4]. Among them are many taxa that are not included in the Crop Wild Relatives lists in many countries, and due to their restricted distribution, they should be given a higher priority for conservation [5]. However, this plant family also includes species that occur in arable lands and that could threaten crop production. Orobanche is the most agriculturally important genera of Orobanchaceae and contains around 200 species distributed all over the world [3]. In Iran, there are 36 different Orobanche speciesthat have been recorded [6]. All plants in the genus Orobanche are root parasites that naturally attach to their host with haustoria formation to extract nutrients [7,8]. Orobanche species have been identified in 58 countries [9]. Despite the widespread distribution of Orobanche members worldwide, yield loss of host crops occurs mainly in the north temperate regions, including East and South Europe, West Asia, and North Africa [7,9,10]. In the Mediterranean area and West Asia, Orobanche members threaten about 16 million ha of arable land, and crop yield losses due to Orobanche spp. attacks range from 5 to 100% [11,12,13,14].
In Iran, as in most other regions, Orobanche aegyptiaca (Pers.) is one of the more widespread species and often attacks summer crops, especially Solanaceae (e.g., eggplant, potato, tobacco and tomato) [6]. Beside the O. aegyptiaca, other important species that are considered for research include: O. ramose (L.) Pomel (which often attacks to annual crops, such as tobacco, tomato, hemp, and cabbage), O. crenata Forssk (which grows on legume crops), O. Cumana Wallr. (which attacks Asteraceae crop plants, such as sunflower) [15], O. gracilis Beck. (which growson Fabaceae plants), O. pancicii Beck. (parasitizing Knautia L. and possibly also Scabiosa L. species), Phelipanche lavandulacea Pomel. (whose single perennial host is Bituminaria bituminosa (L.) C. H. Stirt.), and finally P. purpurea (Jacq.) Soja′k. (which often grows on Asteraceae plants such as Artemisia L. and Achillea wilhelmsii C. Koch) [15,16].
Our understanding of the phylogenetic relationships among major clades of Orobanche [17,18,19], along with species-level relationships, have been greatly advanced by studying molecular data [20,21,22].
Identification of Orobanche species is difficult because herbarium material lose their color and vegetative organs are reduced. Furthermore, the majority of Orobanche species are rare and endangered, and they do not have a clear taxonomic description as a result of their rarity [3].
A species of Orobanche was recorded on spiny cocklebur (Xanthium spinosum L.) for the first time. This study was conducted to make a phylogenetic analysis of this new isolate of the holoparasitic plant belonging to Orobanche genus using the sequences of ITS1-ITS2 region, and to better understand its sequence pattern in the studied rRNA region.

2. Results

2.1. Morphological Characters

The attachment of the collected sample of broomrape (Iranian isolate, IR-Iso) to spiny cocklebur (X. spinosum) roots was verified visually (Figure 1). The stems were erect (14–19 cm height), branched, glandular-pubescent and pale yellowish. The bracts were 0.5 to 0.6 cm long.
The flowers were surrounded by one bract and two bracteoles. The bracts were 0.4 to 0.5 cm long, and bracteoles measured 0.5 cm. The calyces were gamosepalous, 0.4 cm long, and glandular. Corolla were medium slate blue with darker veins, 1.8 to 2.0 cm, conspicuously infundibuliform, slightly curved, and glandular-pubescent. Stamens were epipetalous, inserted 0.5 cm above the corolla base, with hairy filaments, 1.1 to 1.4 cm long, and the anthers were villous. The style (measured 1.4 to 1.6 cm) and stigma (measured 0.5 cm) lobes were light steel blue.

2.2. Sequence Analysis and Identities

5.8S rRNA fragments from the Iranian isolate were amplified using specific primers that bind to conserved regions of the ITS sequences. A unique PCR product of approximately 700 bp was generated (in two samples) and nucleotide sequences of the PCR products were determined by Sanger sequencing (613 nucleotides). Sequences from the two samples were 100% identical. The sequence (hereafter named IR-Iso) spanning the ITS1, 5.8S rRNA and ITS2 regions were aligned to other known Orobanchaceae sequences stored in GenBank. Seventy-one accessions in NCBI showed a significant similarity to IR-Iso. These 71 accessions, plus 11 additional accessions from the work of Frajmanet al. [23], formed our data set. Haplotyping analysis showed that 30 haplotypes (including the IR-Iso) existed in the data set (Table 1; Supplementary File S1). TheIR-Iso differed from most similar GenBank accessions by 5.6–6.7%, but was more distant from the 11 additional accessions in [23] (from number 22 to 30 in Table 1).
The nucleotide frequencies from the sequences of the Orobanchaceae ITS1-ITS2 region in our data set (including out group sequences) are illustrated in Figure 2. It seems that significant differences exist in the frequency of A/T (21.9% vs. 24.2%) and G/C (26.4% vs. 27.4%) nucleotides. Also, G+C content (53.9%) is significantly higher than A+T content (46.1%).
Distance analysis in MEGA6 (Supplementary Table S1) showed that in general IR-Iso showed minimum (5.9%) and maximum (41.3%) sequence divergence to KC811184 (O. aegyptiaca isolate 26-9S163TJGFQ2) and AY209282 (O. anatolica Boiss. &Reut. isolate 1) respectively, with overall divergence estimated at 11.96% (SE = 0.012).

2.3. Genetic Diversity Analysis

Maximum likelihood estimation (MLE) showed that the Kimura 2-parameter model (1980) was the best-fitted model for the nucleotide substitution pattern (Supplementary Table S2). Thus, genetic diversity was performed using the Kimura 2-parameter model.
MCL estimate of the nucleotide substitution pattern showed that transitional substitutions (bold and italicized values in Table 2) were significantly higher than transversional substitutions. The transition/transversion ratios were k1 = 3.69 (purines) and k2 = 5.95 (pyrimidines). The overall transition/transversion (R) bias was equal to 2.44.

2.4. Cluster Analysis

Thirty accessions, including IR-Iso, were classified by the neighbor-joining (NJ) method (Figure 3). Based on the NJ tree, two distinct groups were recognized with bootstrap values higher than 50%. High bootstrap support was obtained for the first group (99%), which contained two clades. Clade I has twenty-one Orobanchaceae species. As seen, most Orobanchaceae species were clustered together, and hence are monophyletic. An exception to this is the Orobanche purpurea (Jacq.), which alone formed a distinct clade in the basal side of the first clade in group I. As seen in Figure 3, the Iranian ecotype IR-Iso is co-clustered with O. aegyptiaca isolates 26-9S163TJGFQ2 from China. The seven accessions placed into group II have six clades.
AMOVA showed that the mean diversity in the entire population was 0.123 (SE = 0.013), which consisted of the inter-population diversity of 0.076 (SE = 0.012) and the diversity within subpopulations of 0.047 (SE = 0.004), forming 61.8% and 38.2% of total diversity, respectively. AMOVA also showed that between-population variation composed a large part of the genetic differentiation (61.9%, SE = 0.04).
The above grouping was confirmed using Bayesian structure analysis (Figure 4). It can be seen that there are two real groups in the studied population, which is revealed by using the ΔK method proposed by Evanno et al. [24] (top graph in Figure 4). The same accessions that form the two groups depicted in Figure 3 were assigned to distinct groups by the Bayesian method of structure analysis (bottom graph in Figure 4).
Tajima’s neutrality test showed that 37.9% of sites were segregated in the data set (Table 3), but only 9.6% of them were phylogenetically informative. Accessions in the data set showed a high similarity, as the overall mean genetic distance (π) in the data set was nearly 8.8%. Tajima’s D was equal to −0.304, indicating no deviance of mutation-genetic drift equilibrium; in other words, there is no evidence that the selection was a powerful force in the evolution of the studied accessions. In contrast, when the neutrality test was completed for group I, which consisted of 22 accessions, it was seen that only 21.8% of sites were segregated,6.9% of which was the nucleotide diversity, indicating the existence of more genetic similarity in this group. However, 25.2% of sites were segregated in group II, only 2.6% of which was the nucleotide diversity in the group.

3. Discussion

The ITS region of the 18S-5.8S-26S nuclear ribosomal cistron is now extensively used around the world for taxonomic classification, having been first utilized more than two decades ago [25,26]. Nuclear rDNA has hundreds to thousands of repeats in plant genomes, therefore, they are more easily isolated than most low-copy nuclear loci. Additionally, little experimental expertise is required for their successful amplification. The isolated sequence of ITS1-ITS2 region in this study contained 613 nucleotides. In general, the length of ITS sequences in plants has a relatively wide range (approximately 500–700 bp in angiosperms [27] to 1500–3700 bp in some gymnosperms [28,29]). It has been reported that this level of ITS sequence variation is suitable for phylogenetic inference at the specific, generic or even family levels [25,27]. Baldwin and other researchers concluded that the variation at hierarchical levels is related mostly to nucleotide polymorphisms, among which indel polymorphisms are common.
In this study, we described the molecular characterization and phylogenetic analysis of the 5.8S rRNA genic region of a new isolate of Orobanche, associated with host weed X. spinosum in one locality of Karaj, Alborz Province, Iran. The ITS sequences were also used to confirm the identity of broomrapes in species Aconitum lycoctonum L. in the Alps [4].
It was supposed that because of the removal of ITS sequences during rRNA transcript processing (e.g., cleavage of the primary transcript within ITS-1 and ITS-2 during maturation of the small subunit, 5.8S, and the large subunit, ribosomal RNAs), they would be subject to mild functional limitations, which in turn would offer a prevalence of nucleotide sites evolving neutrally [30,31].
DNA sequence divergence data, particularly divergence in 5.8S rRNA, are widely used for defining species [19]. The IR-Iso isolate studied here showed 5.8–6.6% divergence from most homologous Orobanche species, and up to 37.9% divergence from distant species (Supplementary Table S1). In our work, the average divergence between the studied species was estimated at 12.0%, with 9.6% of them being phylogenetically informative. Therefore, a cut-off of 12% divergence is recommended as a criterion for demarcating species in this research. Baldwin et al. [27] reported divergence values ranging between 0–39% in pairwise comparisons between taxa, of which 5–59% was potentially phylogenetically informative. DNA sequence comparisons of the ITS1-ITS2 region sequence revealed ~82.7–94.5% identity with known Orobanche spp. and indicated that the Iranian isolate (IR-Iso) was more closely related to O. aegyptiaca, and this was confirmed by using Bayesian structure analysis (Figure 3), although the disparity index test (Supplementary Table S3) showed that IR-Iso has evolved with a different pattern of nucleotide substitution relative to all other known Orobanche species.
Orobanche aegyptiaca is the most agriculturally important species of broomrape in Iran, and it is known to attack important crops and some ornamental plants, such as Kalanchoe blossfeldiana (Poelln.) [32]. However, to our knowledge, this study reports the first occurrence of a spiny cocklebur plant as a host for O. aegyptiaca. Spiny cockleburis an annual weed that is widely dispersed around the world. Our findings suggest that spiny cocklebur plants could serve as a ″green ridge″, allowing O. aegyptiaca to grow and produce large quantities of seeds freely in the absence of other host crops (e.g., in fallow conditions or outside of fields), however, this in turn could increase the infestation level that occurs in the following years. Therefore, control of this wild host plant should be given a greater priority as a part of an integrated weed management system to avoid increasing the weed seed bank in the soil.

4. Materials and Methods

4.1. Plant Material and Sampling

Plant material of Orobanchaceae from Chaharbagh (35°50′20″ N 50°50′53″ E), a locality in Karaj, Alborz Province, Iran (Figure 5), was collected on a summer annual weed plant (X. spinosum).

4.2. Extraction of DNA, PCR Condition and DNASequencing

DNA from leaf samples was extracted using the cetyltrimethyl ammonium bromide (CTAB) method described in [33]. Amplification and sequencing of the given DNA region was completed using the method described by Schneeweiss et al. [19]. DNA sequencing was performed by using an ABI automated sequencer (Bioneer Co., Seoul, Korea).

4.3. Sequence Analyses and Phylogenetic Studies

BLASTn searches of the GenBank ‘nr’ database was used for initial sequence identification. Sequence alignment was achieved using multiple sequence alignment software ClustalW. The data set consisted of 19 sequences from NCBI that showed a significant similarity with our query of sequences 613 nucleotides in length, obtained by using the BLAST tool, and an 11 additional accessions studied by Frajman et al. [23]. The accessions with GenBank numbers in the molecular analysis are given in Table 1. For building the phylogenetic tree, firstly the model selection was performed in MEGA6 software [34]. Subsequently, the model with the lowest Bayesian Information Criterion (BIC) score was considered the best model for the description of the substitution pattern. The evolutionary history was inferred using the neighbor-joining method [35] in MEGA6. A non-parametric bootstrap analysis with 5000 replicates provided quantitative support for recovered nodes [36]. Branches that hadless than 50% bootstrap replicates were collapsed. Distance and pattern analysis tools of MEGA 6.0 software were used for further analysis of the sequences. O. anatolica isolate 1 was used as an outgroup, because it had the longest branch in the preliminary phylogenetic tree.
For the estimation of the substitution matrix, substitution patterns and rates were estimated using the TamuraNei [37] model. The relative instantaneous r values were calculated. An estimation of the pattern of nucleotide substitution and rates was performed usingthe Kimura 2-parameter model [38]. The molecular clock was tested by comparing the ML value for the given topology both with and without the molecular clock constraints usingthe above-mentioned model [38].

5. Conclusions

Based on the molecular features of the given 5.8S rRNA region, such as nucleotide similarity, disparity index test and phylogenetic analysis, it can be concluded that the Iranian isolate is a new separate isolate, as it has clear differences from other analyzed accessions as a whole, and even from the closest isolates of O. aegyptiaca that co-clustered with them. Therefore, it can be considered to bea new ecotype of O. aegyptiaca, because it has the capability of parasitizing a new host, X. spinosum. Considering the host specialty and molecular analyses, we propose IR-Iso is a new ecotype of O. aegyptiaca and suggest its scientific name as O. aegyptiaca ecotype Alborzica. To our knowledge, this is the first report of infestations of O. aegyptiaca on X. spinosum. This relationship can be important from two perspectives. Firstly since Orobanche is an obligate parasite plant, it can only grow in the presence of the host plants, therefore, in arable lands, the presence of X. spinosum, especially during the fallow year (land that is left unseeded during a growing season) can guarantee growth and seed production of O. aegyptiaca. Therefore, the parasite weed could pose a serious threat to crops, its occurrences during the fallow should be monitored and controlled using appropriate weed management practice to minimize the production of new seeds. Second; the presence of X. spinosum as a host plant in wild lands will allow O. aegyptiaca to grow freely, which will prevent the extinction of this species, and help preserve biodiversity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11111406/s1, Table S1, Estimates of evolutionary divergence between sequences; Table S2, Maximum likelihood fits of 24 different models of nucleotide substitution; Table S3, Test of the homogeneity of substitution patterns between sequences. Supplementary file S1, Sequences of Orobanche species.

Author Contributions

Conceptualization, A.R.Y. and A.A.; formal analysis, R.F., L.R. and F.S.; investigation, R.F., L.R. and F.S.; data curation, A.R.Y. and A.M.; writing—original draft preparation, A.R.Y., A.A. and R.F.; writing—review and editing, A.M. and D.L.U.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by University of Zanjan, Iran.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AMOVAAnalysis of molecular variance
BLASTbasic local alignment search tool
CTABcetyltrimethyl ammonium bromide
InDelinsertion-deletion mutations
ITSinternal transcribed spacer
MCLMaximum composite likelihood
MEGAMolecular Evolutionary Genetics Analysis
PCRpolymerase chain reaction. The sequence of targeted region spanning ITS1, 5.8S rRNA and ITS2 was deposited in Gene bank (www.ncbi.nlm.nih.gov) and it’s accessible with accession number MG948171.1.

References

  1. McNeal, J.R.; Bennett, J.R.; Wolfe, A.D.; Mathews, S. Phylogeny and origins of holoparasitism in Orobanchaceae. Am. J. Bot. 2013, 100, 971–983. [Google Scholar] [CrossRef] [PubMed]
  2. Simpson, M.G. Diversity and Classification of Flowering Plants: Eudicots. In Plant Systematics, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 2010; pp. 275–448. [Google Scholar] [CrossRef]
  3. Piwowarczyk, R.; Kwolek, D.; Denysenko, M.; Cygan, M.; Goralski, G.; Slesak, H.; Tuleja, M.; Joachimiak, A.J. Orobanche grenieri (Orobanchaceae), a southwestern European species newly found in Asia. Ann. Bot. Fenn. 2015, 52, 411–418. [Google Scholar] [CrossRef] [Green Version]
  4. Schneeweiss, G.M.; Frajman, B.; Dakskobler, I. Orobanche lycoctoni Rhiner (Orobanchaceae), a poorly known species of the Central European flora. Candollea 2009, 64, 91–99. [Google Scholar]
  5. Perrino, E.V.; Wagensommer, R.P. Crop Wild Relatives (CWRs) Threatened and Endemic to Italy: Urgent Actions for Protection and Use. Biology 2022, 11, 193. [Google Scholar] [CrossRef]
  6. Nosratti, I.; Mobli, A.; Mohammadi, G.; Yousefi, A.R.; Sabeti, P.; Chauhan, B.S. The problem of Orobanche spp. and Phelipanche spp. and their management in Iran. Weed Sci. 2020, 68, 555–564. [Google Scholar] [CrossRef]
  7. Rubiales, D.; Verkleij, J.; Vurro, M.; Murdoch, A.J.; Joel, D.M. Parasitic plant management in sustainable agriculture. Weed Res. 2009, 49, 1–5. [Google Scholar] [CrossRef]
  8. Erdogan, P. Parasitic Plants in Agriculture and Management. In Parasitic Plants; Elsevier: Amsterdam, The Netherlands, 2022. [Google Scholar] [CrossRef]
  9. Habimana, S.; Nduwumuremyi, A.; Chinama, R.J.D. Managementof orobanche in field crops: A review. J. Soil Sci. Plant Nutr. 2014, 14, 43–62. [Google Scholar] [CrossRef]
  10. Ma, Y.; Zhang, W.; Dong, S.; Ren, X.; An, Y.; Lang, M. Induction of seed germination in Orobanche spp. by extracts of traditional Chinese medicinal herbs. Sci. China Life Sci. 2012, 55, 250–260. [Google Scholar] [CrossRef]
  11. Negewo, T.; Ahmed, S.; Tessema, T.; Tana, T. Biological Characteristics, Impacts, and Management of Crenate Broomrape (Orobanche crenata) in Faba Bean (Vicia faba): A Review. Front. Agron. 2022, 4, 708187. [Google Scholar] [CrossRef]
  12. Antonova, T.S.; Araslanova, N.M.; Strelnikov, E.A.; Ramazanova, S.A.; Guchetl, S.Z.; Chelyustnikova, T.A. Distribution of highly virulent races of sunflower broomrape (Orobanche cumana Wallr.) in the southern regions of the Russian Federation. Russ. Agric. Sci. 2013, 39, 46–50. [Google Scholar] [CrossRef]
  13. Fetahaj, R.; Kabashi, B.; Mehmeti, A. Parasitic Plants in Forage Legumes—Medicago sativa L. In Parasitic Plants; Elsevier: Amsterdam, The Netherlands, 2022. [Google Scholar] [CrossRef]
  14. Croes, A.F.; Schapendonk, W.; Jansen, F.; Stommen, P.; Bentsink, L.; Zwanenburg, B.; Wullems, G.J. Germination of Orobanche-Aegyptiaca and O-Crenata Is Stimulated Invitro by Isolated Roots—A Comparative Study. In Biology and Management of Orobanche; KIT Press: Amsterdam, The Netherlands, 1994; pp. 157–168. ISBN 90-6832-093-9. [Google Scholar]
  15. Teryokhin, E.S. Some Biological Peculiarities of Orobanche and Its Host Plants and Their Possible Significance for Control Strategies. In Biology and Management of Orobanche; KIT Press: Amsterdam, The Netherlands, 1994; pp. 94–98. ISBN 90-6832-093-9. [Google Scholar]
  16. Yousefi, A.R.; Jamshidi, K.; Oveisi, M.; Karimojeni, H.; Pouryosef, M. First Report of Orobanche purpurea on Achillea wilhelmsii in Iran. Plant Dis. 2013, 97, 694. [Google Scholar] [CrossRef]
  17. Manen, J.F.; Habashi, C.; Jeanmonod, D.; Park, J.M.; Schneeweiss, G.M. Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences. Mol. Phylogenet. Evol. 2004, 33, 482–500. [Google Scholar] [CrossRef]
  18. Park, J.M.; Manen, J.F.; Schneeweiss, G.M. Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae). Mol. Phylogenet. Evol. 2007, 43, 974–985. [Google Scholar] [CrossRef]
  19. Schneeweiss, G.M.; Colwell, A.; Park, J.M.; Jang, C.G.; Stuessy, T.F. Phylogeny of holoparasitic Orobanche (Orobanchaceae) inferred from nuclear ITS sequences. Mol. Phylogenet. Evol. 2004, 30, 465–478. [Google Scholar] [CrossRef]
  20. Li, X.; Feng, T.; Randle, C.; Schneeweiss, G.M. Phylogenetic Relationships in Orobanchaceae Inferred From Low-Copy Nuclear Genes: Consolidation of Major Clades and Identification of a Novel Position of the Non-photosynthetic Orobanche Clade Sister to All Other Parasitic Orobanchaceae. Front. Plant Sci. 2019, 10, 902. [Google Scholar] [CrossRef] [Green Version]
  21. Bennett, J.R.; Mathews, S. Phylogeny of the parasitic plant family Orobanchaceae inferred from phytochrome A. Am. J. Bot. 2006, 93, 1039–1051. [Google Scholar] [CrossRef] [Green Version]
  22. Gaudeul, M.; Siljak-Yakovlev, S.; Jang, T.S.; Rouhan, G. Reconstructing Species Relationships within the Recently Diversified Genus Odontites Ludw. (Orobanchaceae): Evidence for Extensive Reticulate Evolution. Int. J. Plant Sci. 2018, 179, 1–20. [Google Scholar] [CrossRef]
  23. Frajman, B.; Carlon, L.; Kosachev, P.; Pedraja, O.S.; Schneeweiss, G.M.; Schonswetter, P. Phylogenetic position and taxonomy of the enigmatic Orobanche krylowii (Orobanchaceae), a predominatly Asian species newly found in Albania (SE Europe). Phytotaxa 2013, 137, 1–14. [Google Scholar] [CrossRef] [Green Version]
  24. Evanno, G.; Regnaut, S.; Goudet, J. Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study. Mol. Ecol. 2005, 14, 2611–2620. [Google Scholar] [CrossRef] [Green Version]
  25. Baldwin, B.G. Phylogenetic Utility of the Internal Transcribed Spacers of Nuclear Ribosomal DNA in Plants: An Example from the Compositae. Mol. Phylogenet. Evol. 1992, 1, 3–16. [Google Scholar] [CrossRef]
  26. Baldwin, B.G. Molecular Phylogenetics of Calycadenia (Compositae) Based on Its Sequences of Nuclear Ribosomal DNA—Chromosomal and Morphological Evolution Reexamined. Am. J. Bot. 1993, 80, 222–238. [Google Scholar] [CrossRef]
  27. Baldwin, B.G.; Sanderson, M.J.; Porter, J.M.; Wojciechowski, M.F.; Campbell, C.S.; Donoghue, M.J. The Its Region of Nuclear Ribosomal DNA—A Valuable Source of Evidence on Angiosperm Phylogeny. Ann. Mo. Bot. Gard. 1995, 82, 247–277. [Google Scholar] [CrossRef]
  28. Germano, J.; Klein, A.S. Species-specific nuclear and chloroplast single nucleotide polymorphisms to distinguish Picea glauca, P-mariana and P-rubens. Theor. Appl. Genet. 1999, 99, 37–49. [Google Scholar] [CrossRef]
  29. Maggini, F.; Frediani, M.; Gelati, M.T. Nucleotide sequence of the internal transcribed spacers of ribosomal DNA in Picea abies karst. DNA Seq. 2000, 11, 87–89. [Google Scholar] [CrossRef]
  30. Nashimoto, M.; Ogata, K.; Mishima, Y. In Vitro Sequence-Specific Cleavage in Transcribed Spacer of Mouse Precursor Ribosomal-Rna. J. Biochem. 1988, 103, 992–997. [Google Scholar] [CrossRef]
  31. Van Nues, R.W.; Rientjes, J.M.; van der Sande, C.A.; Zerp, S.F.; Sluiter, C.; Venema, J.; Planta, R.J.; Raue, H.A. Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucleic Acids Res. 1994, 22, 912–919. [Google Scholar] [CrossRef]
  32. Yousefi, A.R.; Soheily, E. First Report of Orobanche aegyptiaca on Kalanchoe blossfeldiana in Iran. Plant Dis. 2014, 98, 1287. [Google Scholar] [CrossRef]
  33. Kalbande, B.B.; Patil, A.S.; Chakrabarty, P.K. An efficient, simple and high throughput protocol for cotton genomic DNA isolation. J. Plant Biochem. Biotechnol. 2016, 25, 437–441. [Google Scholar] [CrossRef]
  34. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef] [Green Version]
  35. Rzhetsky, A.; Nei, M. A Simple Method for Estimating and Testing Minimum-Evolution Trees. Mol. Biol. Evol. 1992, 9, 945–967. [Google Scholar]
  36. Felsenstein, J. Confidence-Limits on Phylogenies—An Approach Using the Bootstrap. Evolution 1985, 39, 783–791. [Google Scholar] [CrossRef]
  37. Tamura, K.; Nei, M. Estimation of the Number of Nucleotide Substitutions in the Control Region of Mitochondrial-DNA in Humans and Chimpanzees. Mol. Biol. Evol. 1993, 10, 512–526. [Google Scholar] [CrossRef]
  38. Kimura, M. A Simple Method for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of Nucleotide-Sequences. J. Mol. Evol. 1980, 16, 111–120. [Google Scholar] [CrossRef]
Plants 11 01406 g001 550
Figure 1. (A) The infection of a spiny cocklebur (Xanthium spinosum L.) by the collected broomrape (Orobanche sp.) from Iran. (B) Orobanche isolate detached from the roots of the host plant.
Figure 1. (A) The infection of a spiny cocklebur (Xanthium spinosum L.) by the collected broomrape (Orobanche sp.) from Iran. (B) Orobanche isolate detached from the roots of the host plant.
Plants 11 01406 g001
Plants 11 01406 g002 550
Figure 2. Nucleotide compositions of the 30 haplotype sequences in data set.
Figure 2. Nucleotide compositions of the 30 haplotype sequences in data set.
Plants 11 01406 g002
Plants 11 01406 g003 550
Figure 3. The unrooted tree of nuclear 5.8S rRNA sequences of Orobanchaceae reconstructed by the neighbor-joining (NJ) method considering O. anatolica as outgroup. Numbers at the nodes represent the bootstrap values over 50%. Iranian isolate (IR-Iso) is shown in green.
Figure 3. The unrooted tree of nuclear 5.8S rRNA sequences of Orobanchaceae reconstructed by the neighbor-joining (NJ) method considering O. anatolica as outgroup. Numbers at the nodes represent the bootstrap values over 50%. Iranian isolate (IR-Iso) is shown in green.
Plants 11 01406 g003
Plants 11 01406 g004 550
Figure 4. Bayesian-based clustering of the same individuals assigned to two groups by Mega 6. Real number of groups determined by Evanno et al. (2005) method (top). Graphical representation of the assignment of each individual to one of two red or green groups (bottom). Numbers in parenthesis on horizontal axis are group numbers determined by Mega 6.
Figure 4. Bayesian-based clustering of the same individuals assigned to two groups by Mega 6. Real number of groups determined by Evanno et al. (2005) method (top). Graphical representation of the assignment of each individual to one of two red or green groups (bottom). Numbers in parenthesis on horizontal axis are group numbers determined by Mega 6.
Plants 11 01406 g004
Plants 11 01406 g005 550
Figure 5. Map showing the region where material was collected in Karaj, Alborz Province, Iran.
Figure 5. Map showing the region where material was collected in Karaj, Alborz Province, Iran.
Plants 11 01406 g005
Table
Table 1. Sequences showing similarity to IR-Iso ecotype after haplotype analysis.
Table 1. Sequences showing similarity to IR-Iso ecotype after haplotype analysis.
IDAccession NumberScientific NameOriginHost PlantIR-ISO
1IR-IsoOrobanche sp.IranSpiny cocklebur100.0
2AY209326Orobanche pulchellaGeorgia-94.46
3AY960742Phelipanche cf. ibericaTurkey 94.30
4EU581788Orobanche gratiosaSpain-94.30
5EU581777Orobanche gratiosaSpain-94.30
6KC811184Orobanche aegyptiacaChinaTomato94.41
7EU581785Orobanche gratiosaSpain-94.14
8EU581775Orobanche gratiosaSpain-94.14
9AY209332Orobanche cf. coelestisTurkey-94.14
10KC811199Orobanche aegyptiacaChinaPumpkin94.24
11KC811218Orobanche aegyptiacaChinaTomato94.08
12KC811209Orobanche aegyptiacaChinaTomato94.08
13KC811208Orobanche aegyptiacaChinaTomato94.08
14KC811183Orobanche aegyptiacaChinaTomato94.08
15KC811180Orobanche aegyptiacaChinaChili94.08
16EU581766Phelipanche sp.Spain-93.81
17KC811177Orobanche aegyptiacaChinaWatermelon93.91
18EU581745Phelipanche sp.Spain-93.65
19KC811166Orobanche aegyptiacaChinaTomato93.77
20EU581721Phelipanche sp.Spain-93.34
21KF359504Orobanche krylowiiRussia-77.37
22EU817103Orobanche lycoctoniSlovenia-77.31
23AY209260Orobanche bartlingiiCroatia-77.07
24AY209240Orobanche gracilisMorocco-76.42
25AY209282Orobanche anatolicaTurkey-93.65
26EU581738Orobanche rosmarinaFrance-76.97
27AY209257Orobanche raddeanaGeorgia-76.26
28AY209234Orobanche cernuaJordan-88.47
29AY209309Orobanche purpureaGermany-76.19
30EU655623Orobanche elatiorFrance-77.13
Table
Table 2. Maximum composite likelihood estimate of the pattern of nucleotide substitution.
Table 2. Maximum composite likelihood estimate of the pattern of nucleotide substitution.
ATCG
A-3.514.0514.26
T3.16-24.093.87
C3.1620.87-3.87
G11.633.514.05-
Note: Each entry shows the probability of substitution (r) from one base (row) to another base (column) [17]. For simplicity, the sum of r values is made equal to 100. Rates of different transitional substitutions are shown in bold and those of transversional substitutions are shown in italics.
Table
Table 3. Tajima’s test statistics for the 5.8S rRNAplus ITS sequences of Orobanche.
Table 3. Tajima’s test statistics for the 5.8S rRNAplus ITS sequences of Orobanche.
mnSpsθπD
Group I85791260.2180.0840.069−0.945
Group II225841470.2520.0690.026−2.556
Overall305602120.37910.0960.088−0.304
m = number of sequences; n = total number of sites; S = Number of segregating sites; ps = S/n; θ (functional coefficient of divergence) = ps/α1; π = nucleotide diversity; D = the Tajima’s test statistic (Tajima, 1989).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Yousefi, A.R.; Ahmadikhah, A.; Fotovat, R.; Rohani, L.; Soheily, F.; Uberti, D.L.; Mastinu, A. Molecular Characterization of a New Ecotype of Holoparasitic Plant Orobanche L. on Host Weed Xanthium spinosum L. Plants 2022, 11, 1406. https://doi.org/10.3390/plants11111406

AMA Style

Yousefi AR, Ahmadikhah A, Fotovat R, Rohani L, Soheily F, Uberti DL, Mastinu A. Molecular Characterization of a New Ecotype of Holoparasitic Plant Orobanche L. on Host Weed Xanthium spinosum L. Plants. 2022; 11(11):1406. https://doi.org/10.3390/plants11111406

Chicago/Turabian Style

Yousefi, Ali Reza, Asadollah Ahmadikhah, Reza Fotovat, Leila Rohani, Foad Soheily, Daniela Letizia Uberti, and Andrea Mastinu. 2022. "Molecular Characterization of a New Ecotype of Holoparasitic Plant Orobanche L. on Host Weed Xanthium spinosum L." Plants 11, no. 11: 1406. https://doi.org/10.3390/plants11111406

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop