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Article

Application of SRAP Markers to Identify Gender and Species in Genus Ephedra Tourn. ex L.

by
Najla A. Al Shaye
1,*,
Wafaa M. Amer
2,
Mahmoud O. Hassan
3,
Nasr H. Gomaa
3 and
Maha H. Khalaf
3,*
1
Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University, Riyadh 11671, Saudi Arabia
2
Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt
3
Department of Botany and Microbiology, Faculty of Science, Beni-Suef University, Beni-Suef 62521, Egypt
*
Authors to whom correspondence should be addressed.
Diversity 2025, 17(9), 600; https://doi.org/10.3390/d17090600
Submission received: 18 July 2025 / Revised: 17 August 2025 / Accepted: 22 August 2025 / Published: 26 August 2025
(This article belongs to the Section Plant Diversity)
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Abstract

Background: The genus Ephedra (Ephedraceae) is a dioecious gymnosperm, where female individuals produce the pharmacologically active ephedrine alkaloids. Identifying the sex of specimens without reproductive cones is challenging due to their xeromorphic and morphological similarity. The challenges in sex identification complicate conservation and propagation efforts. Methods: Sequence-Related Amplified Polymorphism (SRAP) markers were applied to distinguish genders in five Ephedra species, particularly the vegetative branches, as well as powdered and fragmented specimens. The fresh material for the five studied Ephedra species and two sexes per species (totaling 10 samples; 5 females & 5 males) was collected from Sinai, Egypt. Results: The SRAP marker results revealed an exclusively male-specific band, and this is not applicable in females in the studied species. The applied SRAP markers grouped males and females in different UPGMA clusters and proved their efficiency in distinguishing between males and females in the five studied species. The Polymorphic Information Content (PIC) values are low (0.16–0.38); this suggests moderate genetic diversity between the females of the studied species, reflecting slow evolutionary rates. Conclusions: The SRAP markers are efficient for identifying Ephedra species at the species and gender levels, even in the absence of sex organs and molecular sequences. Recommendation: This study recommends the use of SRAP markers for conserving and propagating female plants for ephedrine production and suggests sequencing a 95 bp male-specific band to determine if it corresponds to a known sex-linked gene.

Graphical Abstract

1. Introduction

Gymnospermae consists of 14 families and 873 species worldwide [1]. In the Egyptian flora, only the families Cupressaceae and Ephedraceae are represented [2]. Globally, this genus comprises 73 recognized species (POWO, retrieved 8 January 2025). Ephedra species are found across both the Old and New Worlds [3,4]. These plants are typically perennial, dioecious shrubs or under-shrubs [5], characterized by opposite or whorled leaves that are often reduced to membranous sheaths [6]. They display xeromorphic traits and have assimilating branches. The identification of Ephedra species and sex is mainly based on the features of the reproductive cones in both male and female individuals, where female flowers are solitary or in clusters of 2–3, supported by 2–4 pairs of bracts, with ovules enclosed by fleshy or scarious bracts. Male flowers are subtended by a bract, a two-lipped perianth, and a staminal column bearing 2–9 anthers [7].
The genus Ephedra is notably understudied regarding sex identification among gymnosperms [7,8]. The sex identity is a crucial issue for the identification of Ephedra to the section level (section Asarca Stapf, section Alatae Stapf, and section Ephedra [9].
The genus Ephedra in the Egyptian flora has five species belonging to two sections (Alatae and Ephedra). Section Alatae includes E. alata, while section Ephedra comprises E. pachyclada subsp. Sinaica, E. aphylla, E. ciliata, and E. foemina [6,8]. According to the IUCN Red List, E. alata and E. pachyclada are classified as “Least Concern” species [7].
The genus Ephedra is a significant source of pharmacological and environmental potential. However, identifying Ephedra species remains taxonomically challenging, particularly for specimens lacking reproductive cones or material in a powdered form. This difficulty arises from their xeromorphic characteristics, such as reduced leaves and a resemblance to photosynthetic branches [7]. Rydin et al. [9] noted that Ephedra species share highly similar morphological traits. This genus is generally considered monophyletic [10,11,12]. This study introduces the SRAP markers as an alternative approach to identify the fragments/powdered Ephedra specimens at the sex level.
Ephedra spp. are of global importance; despite their economic and pharmacological importance, molecular studies aimed at distinguishing male and female individuals remain limited. To date, the molecular investigations have been restricted to a transcriptome analysis of Ephedra sinica to identify the differentially expressed genes (DEGs) between male and female plants, revealing potential sex-biased metabolic pathways [13]. Ephedra might possess early-stage sex chromosomes or sex-determining regions [14,15,16], but no definitive molecular markers have been validated. Plastid and nuclear DNA sequences across Ephedra species [17] do not focus on sex-linked polymorphisms. Ickert-Bond et al. [18] explored phylogenetic relationships using ribosomal DNA; nonetheless, sex differentiation was not a primary focus. Finally, epigenetic mechanisms (e.g., DNA methylation) that may regulate sex expression in Ephedra, akin to other gymnosperms, were studied [14,19,20], but experimental validation is lacking.
However, female plants are the primary source of ephedrine alkaloids [10,21], whereas male plants contain only trace amounts of these compounds [7], making sex identification crucial for cultivation, conservation, and medicinal use. The international molecular database was lacking molecular information for the studied species at the sex level (National Center for Biotechnology Information (NCBI) https://www.ncbi.nlm.nih.gov/nuccore/?term=Ephedra, accessed on 12 August 2025).
Fortunately, several molecular tools have been used to distinguish between male and female individuals in dioecious plant species. These include SSR (Simple Sequence Repeat) [22], SCAR (Sequence Characterized Amplified Region) [23], and SRAP (Sequence-Related Amplified Polymorphism) [24]. Wu et al. [16] reported that SSR is ideal for population genetics and requires prior genomic data; SCAR is dependent on initial polymorphism screening [18], while SRAP targets the open reading frames, useful for functional gene diversity [14]. Unlike SSR and SCAR, SRAP markers do not require prior DNA sequence information [25]. SRAP markers are also reproducible, cost-effective, and efficient at distinguishing genders in dioecious species [24,25,26,27].
To date, universal molecular markers (markers linked to all the Ephedra species) to reliably distinguish between Ephedra sexes are lacking. This study introduces SRAP markers as an alternative approach for sex identification in Ephedra species. The study objectives are (1) demonstrate the applicability of SRAP markers for distinguishing between genders in dioecious gymnosperms (using Ephedra as a case study), (2) identify sex of Ephedra species in the absence of reproductive cones, and (3) provide an efficient tool for identifying the sex of Ephedra species when specimens are provided as powder or fragments for pharmacological purposes.

2. Materials and Methods

2.1. Sampling and Study Area

Five Ephedra species (E. alata Decne., E. aphylla Forssk., E. ciliata Fisch. and C.A. Mey., E. foeminea Forssk, and E. pachyclada Boiss.) were represented by ten samples/species (5 male and 5 female/species). Juvenile vegetative branches were collected from the current year (2023) growth, from one population to nullify the environmental effect from their localities at mountain cliffs and Wadis (Table 1) in South Sinai. These species, including their distribution range, morphological, taxonomical, and anatomical features, were recognized in the earlier studies [2,3,4,26] in addition to the online references, namely, POWO, 2025 (https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:328160-2, accessed on 19 January 2025, WCVP (The World Checklist of Vascular Plants, accessed on 20 January 2025), IPNI (https://www.ipni.org, accessed on 15 January 2025), and the PLANT LIST (http://www.theplantlist.org/browse/G/Ephedraceae/Ephedra/, accessed on 15 January 2025). Voucher specimens were deposited in Cairo University Herbarium CAI), where samples were arranged according to the Engler System without numbers.

2.2. Genomic DNA Extraction and Amplification

Genomic DNA was extracted from 30 cm apical juvenile branches. Five samples/gender for each species were tested as replicates, and each was pulverized (10 specimens/species and 5/sex). The branches were homogenized using liquid nitrogen, and DNA isolation was performed using the modified CTAB method [25], where 0.5 mL of 2× CTAB buffer was added and incubated at 65 °C for 90 min. DNA was precipitated by 2-propanol, washed with 70% ethanol, and dissolved in TE buffer.
For genomic DNA amplification, the first five cycles of the PCR process are conducted for 1.0 min/each of the following stages with the following temperatures: 94 °C for denaturation, 35 °C for annealing, and 72 °C for extension. After these initial cycles, the annealing temperature is increased to 50 °C for an additional 35 cycles. For the amplification, we utilized the routine procedure adopted for PCR markers [28]. This resulted in separation by acrylamide gels and detection through autoradiography.

2.3. Sequencing of SRAP Marker Bands

For sequencing the developed Ephedra species bands, a protocol to isolate DNA from SRAP gels for direct sequencing was developed according to Li et al. [25] as follows: denaturing thick polyacrylamide gels (size 35 × 43 cm, thickness 0.8 mm) were prepared, then 20 µL/well was loaded for each tested sample. After electrophoresis, the gel was exposed overnight to a high-sensitivity film (Kodak BioMax). Using the exposed film as a guide, the gel pieces containing the polymorphic bands were cut and placed into a dialysis tube. This dialysis tube was then positioned in the buffer tank of a sequencing-gel apparatus, where the DNA was electroeluted in 1× TBE buffer (Fisher FB-SEQ 3545), at 2000 V. After DNA precipitation by ethanol and suspension in TE buffer, the DNA was sequenced using an ABI377 sequencer (ADWIC, Cairo, Egypt).

2.4. SRAP Analysis

Nine positive primer pairs out of 15 (forward and reverse) were used in various combinations (Table 2). The SRAP reaction mixture and protocols were applied according to Ferriol et al. [29]. Each 25 µL PCR reaction contained 20 ng genomic DNA, 200 µM dNTPs, 1.5 mM MgCl2, 0.3 µM primer, 10× Taq buffer, and 1 unit of Fermentas Taq polymerase. Amplification was performed in an Eppendorf Mastercycler Gradient with the following profile: 5 cycles of denaturation at 94 °C for 1 min, annealing at 35 °C for 1 min, and elongation at 72 °C for 2 min. Amplified products were separated by electrophoresis on a 2% agarose gel in 0.5× TBE buffer, stained with ethidium bromide, and visualized under UV light [30].

2.5. Statistical Analysis

Genetic similarity between species was calculated using Jaccard’s coefficient in SPSS (version 20). A dendrogram was generated using UPGMA cluster analysis in PAST (version 3.26). The Dice coefficient (GSij = 2a/(2a + b + c)) was also applied [31]. Hierarchical clustering (heatmap) was performed in R (version 4.3.2) using the “pheatmap” and “RColorBrewer” packages.

3. Results and Discussion

3.1. Morphometric Features

Ephedra species are woody sub-shrubs, drought-resistant, with stems not exceeding 4 m in height, densely branched, and with green branchlets, assimilating opposite or in whorls with scale-like, deciduous leaves (Figure 1). Ephedra species are dioecious; the male and female reproductive organs are arranged in cones carried in different individuals, with each cone subtended by 2–4 or more pairs of scale-like opposite bracts. But the lack of these reproductive cones hinders taxonomists from distinguishing sexes/species due to the analogous morphometric features of the assimilating branches across all the species, including sex (Figure 1) [7].
Figure 1 highlights the morphological similarities in the five studied Ephedra spp., which complicates their taxonomic identity up to the sex level in the absence of reproductive cones. This aspect is general in dioecy species and is found in almost 65% of the Gymnosperm species [24,32]. Figure 1B–G show the close morphometric similarities between the studied five species; additionally, there are no morphometric differences between male and female vegetative branches of the same species. Nevertheless, the application of stem anatomy was an efficient tool to distinguish between male and female Ephedra species [33]. We still have a gap in the identification of fragmented and powdered material used for pharmacological studies/products, in addition to the identification of juvenile individuals for conservation and propagation programs. Therefore, the Sequence-Related Amplified Polymorphism (SRAP) markers may play a role in filling this gap in our knowledge.
Diversity 17 00600 g001
Figure 1. (A) Xeromorphic habit of the Ephedra spp. in the field; (B) enlarged branches, and assimilating branches showing reduced leaves of the studied species; (C) E. alata; (D) E. aphylla; (E) E. ciliata; (F) E. foemina; and (G) E. pachyclada. These are original photographs taken for this manuscript, but pictures (C,F) are from Khalaf et al. [7,33].
Figure 1. (A) Xeromorphic habit of the Ephedra spp. in the field; (B) enlarged branches, and assimilating branches showing reduced leaves of the studied species; (C) E. alata; (D) E. aphylla; (E) E. ciliata; (F) E. foemina; and (G) E. pachyclada. These are original photographs taken for this manuscript, but pictures (C,F) are from Khalaf et al. [7,33].
Diversity 17 00600 g001

3.2. SRAP Markers for Molecular Characterization of Ephedra Species/Genders

To date, information about the genome sequence of the Ephedra species is still lacking, and at least an attempt to identify males and females of all Ephedra species is also lacking (NCBI, https://www.ncbi.nlm.nih.gov/nuccore/?term=Ephedra, accessed on 20 January 2025). The application of SRAP in this study offers an alternative that may provide positive evidence to distinguish between male and female powdered specimens. The SRAP markers results (Supplementary Table S1) showed universal bands, e.g., 75 bp in EM1/ME6, 55 bp in EM3/ME8, and 85 bp in EM8/ME9 are characteristic of all the studied Ephedra species and are present in all male and female specimens, indicating they are specific bands to the genus Ephedra.
The generated UPGMA dendrogram (Figure 2) based on SRAP results (Supplementary Table S1) illustrated that the ten studied Ephedra genders (2 genders/species) fall into two clusters. Cluster I grouped the female genders, and Cluster II grouped the male genders of the studied Ephedra species. Notably, the fatty acid composition [7] and stem anatomy were shown to be relevant results and considered among the efficient taxonomic tools in the absence of the reproductive organs for the identification of gender in Ephedra species [33]. This grouping is attributed to the male-specific and male-related bands grouping all males across the studied species: primer EM6/ME10, as well as band at 95 bp present in males of all species and absent in females of all species; this band distinguishes male from female genders in the studied Ephedra species, and it is considered a male-specific band. Other bands are present in males of most of the studied Ephedra species but not exclusively in all males, for example, primer EM1/ME6, as well as the band at 145 bp present in males of alata, aphylla, ciliata, foemina, and pachyclada but also present in females of aphylla and ciliata (these points need further study). Moreover, E. alata and E. aphylla are distinguished by the presence of exclusively female-specific (EM8/ME2 at 85 bp and 70 bp). Unfortunately, no detailed sex-linked data are available for Ephedra species, but a relevant male-specific band for Me1/Em5 (130 bp) was detected by Li et al. [34] in Carica papaya (Caricaceae).
On the other hand, there are no bands specific to females (F) in all the studied Ephedra species. However, some bands are present but not exclusively in all females, for example, primer EM8/ME2, as well as the band at 85 bp, present in females of two species (E. alata and E. aphylla) and absent in males of all species; and primer EM3/ME8, as well as the band at 85 bp present in females of alata, aphylla, ciliata, foemina, and pachyclada but also present in males of three of them (aphylla, ciliata, and pachyclada). Moreover, E. alata and E. aphylla are distinguished by the presence of exclusively female-specific (EM8/ME2 at 85 bp and 70 bp). However, the female-specific bands are not commonly detected in any of the Gymnospermae. In Angiospermae, a female-specific band for ME7/EM4 (250 bp) was detected during the early sex identification of Populus cathayana (Salicaceae) by Wang et al. [35]. Similarly, a band at 240 bp for ME9/EM2, in Buchole dactyloides (Poaceae), was detected by Zhou et al. [26].
The aim of this study was largely achieved by this dendrogram, confirming that the SRAP markers were able to distinguish and separate male from female genders, each in a distinct cluster (Figure 2). Hajari et al. [24] achieved congruent results using SRAP markers, where they successfully differentiated male and female genders of Ephedra ciliate using EM3ME7 and EM8ME9 markers, which produced gender-specific bands at 95 bp in females and 85 bp in males, respectively. SRAP markers have proven to be appropriate for gender determination in dioecy species belonging to different Angiospermae families. Zhou et al. [26] distinguished female genders of Buchole dactyloides (Poaceae) via a female-specific band at 240 bp for ME9/EM2; Zheng et al. [36] distinguished a female Ficus awkeotsang (Moraceae) via a band at 240 bp for ME5/EM14; Wu et al. [37] obtained a similar result in Ficus pumila (Moraceae) using three markers ME1/EM2/EM14; and Kumar and Agrawal [38] used it to identify male individuals in Tricosanthes dioica (Cucurbitaceae) by male-specific bands (at 230 and 290 bp for ME4/EM6).

3.3. SRAP-Based Similarity Indexes (SIs) Between the Inter- and Intraspecific Studied Species

The interspecies similarities (different species, as well as the same or different genders; Table 3) for the studied species showed its strong similarity (0.96) with the following pairs: E. aphylla male and E. ciliata female, and E. aphylla male and E. ciliata male, while the minimum similarities (0.72) were recorded between the following pairs: E. foemina male and E. aphylla female, and E. pachyclada male and E. aphylla female. The intraspecies similarity (i.e., within the same species irrespective of genders; Table 3) revealed that the maximum similarity (0.92) was recorded for E. ciliata genders, followed by E. aphylla (0.88), while the lowest similarity (0.81) was observed from both E. foemina and E. pachyclada.
The similarity outlined in Table 3, based on SRAP results, reflects its reliability for gender identification. The SRAP polymorphism rate was 89% in Ephedra ciliata, from the family Ephedraceae, gymnospermae [24], while the accuracy reached 92% in distinguishing genders in juvenile Actinidia chinensis plants from the family Actinidiaceae, angiospermae [26].
The observed highest similarities were between male of E. alata, E. aphylla, and E. ciliata, while the lowest similarities were observed between E. aphylla female and male of both E. foemina and E. pachyclada (Table 3), consistent with the previous studies by Khalaf et al. [7,33] for the taxonomic features, anatomical structure, and fatty acid compositions carried out on the same species to the gender level.

3.4. Heat Map Showing the Genetic Variations of Genders at Inter- and Intra-Specific Levels

3.4.1. Male Genders

The constructed heat map (Figure 3), based on SRAP markers that represent the genetic variations of the studied male Ephedra, visualized by color intensity, is divided into two clusters. The first cluster includes male individuals of E. ciliata and E. aphylla, with E. alata males grouped with this cluster because they show higher percentages of polymorphism/primer (100% p/p) for EM3ME7, EM4ME6, EM4ME7, and EM6ME10. Although male individuals of E. aphylla and E. ciliata have many total amplified bands (3 T.A.B) of EM8ME9 (Supplementary Table S1), the second cluster groups male individuals of E. pachyclada and E. foemina, which display the highest percentages of polymorphism/primer (100% p/p) for EM4ME7, EM8ME4, and EM6ME10, along with the large number of total amplified bands (1–2 T.A.B) of EM8ME9 and a high percentage of polymorphism/primer (ranging from 50 to 100% p/p) for EM8ME9.
The heat map grouping is congruent with the grouping achieved based on the morphological features of the same species, using the features of the reproductive cones [7,33].

3.4.2. Female Genders

The constructed heat map (Figure 4), based on SRAP markers illustrating the genetic variations of the studied female individuals of Ephedra and visualized by color intensity, groups them into two clusters. The first cluster includes female E. foemina and E. pachyclada, in addition to female E. ciliata, as they have higher percentages of polymorphism per primer (100% p/p) for EM8ME4, EM3ME7, and EM4ME7. The second cluster groups the female E. aphylla and E. alata due to their higher percentages of polymorphism per primer (100% p/p) for EM4ME7, EM3ME7, and EM8ME4. Congruent grouping was observed for the species of E. alata and E. aphylla based on their morphological features [7].

3.4.3. Polymorphic Information Content (PIC), Compared with Other Gymnosperms

Table 4 showed that the Polymorphic Information Content (PIC) values are generally low (0.16–0.38); this suggests moderate genetic diversity between the females of the studied Ephedra species, while other gymnosperms like pines are highly polymorphic (PIC ranging from 0.50 to 0.90) on the application of SSR markers in Pinus taeda [39]. Other slow-evolving species are aligned with Ephedra like Ginkgo biloba (PIC values of 0.25–0.35) using the SNP markers [40]. And Taxus baccata (yew) is comparable to the lower range (PIC values around 0.20–0.30), unlike Ephedra, using AFLP markers [41], indicating that Ephedra species are comparable to the gymnosperms with slow evolutionary rates.

3.4.4. Marker Index (MI) Compared with Other Gymnosperms

Table 4 showed the relatively low Marker Index (MI) values (ranging from 0.0018 to 0.03) of Ephedra are likely due to the moderate PIC and variable multiplex ratios (E). This contrasts with gymnosperms like Picea abies (Norway spruce), where MI ranges from 0.05–0.15, using the SSR markers [42,43]. But the Ephedra MI was aligned with less polymorphic species like Cycas revoluta, where the ISSR markers showed MI values of 0.02–0.04 [44].

4. Conclusions

The studied Ephedra species are among the threatened dioecious species, particularly those affected by climate change, as they thrive in open, arid, or semi-arid habitats. Female Ephedra plants are the primary source of pharmacologically active ephedrine alkaloids. Additionally, over-collection from wild habitats has been observed (Wafaa Amer, field observation). Therefore, conservation and propagation efforts for female populations are highly requested. This request requires accurate gender identification. The aim of this study was largely achieved by SRAP markers that were able to distinguish between and separate male from female genders. Accordingly, SRAP markers can support the conservation and propagation efforts for female populations threatened by climate change and as a primary source of pharmacologically active ephedrine alkaloids.

5. Recommendation

This study recommends sequencing the 95 bp band, as this could help identify whether it corresponds to a known sex-linked gene. Additionally, testing more SRAP primers and applications on all the Ephedra spp. is suggested to validate a sufficient number of universal and sex-specific bands/primers for Ephedra species.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d17090600/s1, Table S1: Data were retrieved from the SRAP primers for the studied Ephedra Species.

Author Contributions

M.H.K. collected the plant specimens, performed the practical work, and prepared the manuscript. N.A.A.S. covered the publication fees and participated in the preparation of the manuscript. M.O.H. supervised the fieldwork. N.H.G. revised the manuscript and supervised the fieldwork. W.M.A. proposed the thesis idea, supervised the practical work, and refined the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R187), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We would like to thank and acknowledge Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia for the support given to this research under project number (PNURSP2025R187).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
No. of T.A.B. Number of total amplified bands
No of P.B.Number of polymorphic bands
No of U.B.Number of unique bands
% p/p% of polymorphism/primer

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Figure 2. UPGMA cluster for male and female individuals of the studied Ephedra species.
Figure 2. UPGMA cluster for male and female individuals of the studied Ephedra species.
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Figure 3. Hierarchical clustering analysis of male genders of the studied Ephedra species based on data retrieved from the SRAP markers. Data are represented by the means of at least five replicates.
Figure 3. Hierarchical clustering analysis of male genders of the studied Ephedra species based on data retrieved from the SRAP markers. Data are represented by the means of at least five replicates.
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Figure 4. Hierarchical clustering analysis of female genders of the studied Ephedra species based on data retrieved from the SRAP markers. Data are represented by the means of at least five replicates.
Figure 4. Hierarchical clustering analysis of female genders of the studied Ephedra species based on data retrieved from the SRAP markers. Data are represented by the means of at least five replicates.
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Table 1. Locations of the study Ephedra samples in South Sinai.
Table 1. Locations of the study Ephedra samples in South Sinai.
SpeciesSexLocalityGPS CoordinatesDate of Collection
NE
E. alataMaleAbo Zeinema, plain29°02′31″33°06′30″14 June 2023
FemaleWadi bed of Wadi
Feiran 		28°43′06″33°37′06″15 May 2023
E. aphyllaFemale and
Male		Wadi bed of wadi
Al-Arbaeen 		28°55′58″33°94′58″17 August 2023
E. ciliataMaleWadi bed of wadi Lethi, 28°33′07″33°58′24″15 July 2023
FemaleMonastery at St.
Catherine 		28°33′25″33°58′23″25 August 2023
E. foeminaFemale and
Male		Gabal Mousa, rocky ridge 28°54′13″33°57′55″16 July 2023
E. pachycladaFemaleAbu Walia, Rocky Ridge28°53′55″33°51′39″16 August 2023
MaleAbu Walia, Rocky Ridge28°53′60″33°50′40″16 August 2023
Table 2. The positive primers were used to distinguish Ephedra species at the gender level.
Table 2. The positive primers were used to distinguish Ephedra species at the gender level.
Primer CodePrimer SequenceMolecular Weight bp
EM1ME6Em-1: GACTGCGTACGAATTAAT
Me-6: TGA GTC CAA ACC GGA CA 		75–145
EM3ME7Em-3: GACTGCGTACGAATTGAC
Me-7: TGA GTC CAA ACC GGA CG 		100
EM3ME8Em-3: GACTGCGTACGAATTGAC
Me-8: TGA GTC CTT TCC GGT GC		55–85
EM4ME6Em-4: GACTGCGTACGAATTTGA
Me-6: TGA GTC CAA ACC GGA CA		120
EM4ME7Em-4: GACTGCGTACGAATTTGA
Me-7: TGA GTC CAA ACC GGA CG		75–90
EM6ME10Em-6: GACTGCGTACGAATTGCA
Me-10: TGAGTCCAAACCGGTGC		95
EM8ME2Em-8: GACTGCGTACGAATTCAC
Me-4: TGAGTCCAAACCGGACC		70–85
EM8ME4Em-8: GACTGCGTACGAATTCAC
Me-4: TGAGTCCAAACCGGACC		95
EM8ME9Em-8: GACTGCGTACGAATTCAC
Me-9: TGAGTCCAAACCGGTCC		85–240
Table 3. Similarity Index (SI) based on the SRAP data for the studied Ephedra Species.
Table 3. Similarity Index (SI) based on the SRAP data for the studied Ephedra Species.
Species and
Genders		E.
alata		E.
aphylla		E.
ciliata		E.
foemina		E.
pachyclada
FemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMale
E. alatafemale1.0
male0.851.0
E. aphyllafemale0.760.831.0
male0.810.960.881.0
E. ciliatafemale0.850.910.830.961.0
male0.780.920.840.960.921.0
E. foeminafemale0.850.910.750.880.910.921.0
male0.840.810.720.780.810.830.811.0
E.
pachyclada		female0.760.910.750.880.910.920.910.811.0
male0.840.810.720.780.810.830.900.820.811.0
Table 4. List of information on SRAP primers for the studied Ephedra Species.
Table 4. List of information on SRAP primers for the studied Ephedra Species.
IndexValue
EM1ME6EM3ME7EM3ME8EM4ME6EM4ME7EM6ME10EM8ME2EM8ME4EM8ME9
H0.320.180.380.320.180.50.180.180.42
PIC0.270.160.300.270.160.380.160.160.33
E1.60.91.50.81.80.50.20.92.1
Hav0.0160.0180.0190.0320.0090.050.0090.0180.014
MI0.0260.0160.0280.0260.0160.0250.00180.0160.03
D0.370.20.450.380.190.780.990.20.52
R0.80.210.40.410.40.21
(H) heterozygocity index, (PIC) Polymorphic Information Content, (E) effective multiplex ratio, (Hav) arithmetic mean of H, (MI) Marker Index, (D) discriminating power, and (R) resolving power.
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Shaye, N.A.A.; Amer, W.M.; Hassan, M.O.; Gomaa, N.H.; Khalaf, M.H. Application of SRAP Markers to Identify Gender and Species in Genus Ephedra Tourn. ex L. Diversity 2025, 17, 600. https://doi.org/10.3390/d17090600

AMA Style

Shaye NAA, Amer WM, Hassan MO, Gomaa NH, Khalaf MH. Application of SRAP Markers to Identify Gender and Species in Genus Ephedra Tourn. ex L. Diversity. 2025; 17(9):600. https://doi.org/10.3390/d17090600

Chicago/Turabian Style

Shaye, Najla A. Al, Wafaa M. Amer, Mahmoud O. Hassan, Nasr H. Gomaa, and Maha H. Khalaf. 2025. "Application of SRAP Markers to Identify Gender and Species in Genus Ephedra Tourn. ex L." Diversity 17, no. 9: 600. https://doi.org/10.3390/d17090600

APA Style

Shaye, N. A. A., Amer, W. M., Hassan, M. O., Gomaa, N. H., & Khalaf, M. H. (2025). Application of SRAP Markers to Identify Gender and Species in Genus Ephedra Tourn. ex L. Diversity, 17(9), 600. https://doi.org/10.3390/d17090600

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