1. Introduction
Halophytes are salt-tolerant plants that grow naturally in saline habitats, lowland coastal flats, inland salt marshes, and seashores which constitute about 7–10% of the world’s land area [
1]. They are naturally adapted to large areas of salt-affected rangelands [
2], often creep, and act as sand binders [
3], thus significantly protecting saline habitats and maintaining ecological stability. Also, various palatable halophytic species like
Atriplex spp.,
Suaeda foliosa, and
Distichlis spp. which grow well in highly saline soil around the world can replace traditional crops as potential sources of fodder [
4] and can serve as reserves to fill the gap of annual fodder storage within the grazing management scheme [
5]. Among various halophytic palatable species around the globe
, Aeluropus lagopoides (L.) Thwaites is a perennial salt-secreting halophytic grass extended along North Africa, the Middle East, the Arabian Peninsula, and Central Asia. The plant largely inhabits coastal areas, inland salt marshes, sabkha edges, or saline soil around cultivated areas [
6,
7]. It also occurs in some inundated coastal habitats of eastern and southern and in the saline arid inland sabkhas of the central and northern regions of Saudi Arabia [
8,
9]. Due to its value as a forage plant [
10] and its ability to prevent soil erosion by forming a dense, vigorous root network [
7,
11],
A. lagopoides attracts the attention of researchers. As a perennial grass,
A. lagopoides is considered a good candidate for salt-affected agriculture lands [
10], and it could be integrated into sustainable landscaping of urban green areas in arid regions [
3].
A. lagopoides mainly propagates as mono-specific stands through rhizomes or colonizes open niches through seeds [
12]. The plant has various functional traits that enable its resistance to harsh environmental conditions, including vegetative propagation via stolon and rhizomes, excessive seed production, strong root system, small leaves, epicuticular wax, and salt-secreting glands [
13].
Different coastal and inland regions of Saudi Arabia exhibit considerable variations in environmental conditions in terms of soil moisture, salinity, light, and temperature [
14,
15]. Usually,
A. lagopoides populations in coastal habitats are subject to harsher environmental conditions compared to populations in inland sabkhas; such environmental stresses have a negative effect on
A. lagopoides seed germination and in consequence its colonization [
12,
16]. Due to its overexploitation such as intense grazing in summer with harsh environmental conditions, this plant is under tremendous stress. As such, adaptive plasticity and vegetative propagation are the only means to maintain the population of
A. lagopoides under such stressful environmental conditions [
12,
17]; however, these approaches could decrease its fitness and genetic diversity [
18].
Genetic resources are among the most treasured resources of any country. Genetic variability present among/within the population of species or individuals is the fundamental element of biological polymorphism and species diversity [
19]. The genetic diversity of species forms the main basis of the adaptation and evolution of species to different environments [
20,
21]. Genetic variability helps to build up the rich gene pool of the species that show heterozygosity in their tolerance/resistance to both biotic and abiotic stress [
22]. Studying genetic diversity in plant populations has significance from different dimensions. Determining the genetic diversity of any plant population helps researchers in managing, collecting, conserving, maintaining, and specifying the plants as well as their usage [
23]. Several methods are employed to assess genetic diversity, where morphological trait measurement is the most commonly used index for simple quantification of genetic variation and an assessment of genotypic performance under normal growing climatic conditions [
24]. However, when plant populations are growing under different environmental conditions, it is difficult to use phenotypic variations to assess genetic variation [
25]. Molecular markers have become a common method of determining genetic diversity in plants growing under different environmental conditions. In scientific research, the application of molecular markers has created new opportunities for identifying and manipulating specific genes. Molecular markers have become increasingly significant in assessing species diversity and evolutionary relationships. DNA-based PCR molecular markers provide higher polymorphism and are not prone to environmental influence [
26,
27].
Halophytes and their habitats attract the attention of nature conservationists, particularly in Central Europe [
28]. However, few studies have used molecular markers to assess the genetic variability of halophytes between different habitats [
29,
30]. Recently, using a combination of ISSR-AFLPs,
Aeluropus ecotypes have shown significant variation [
31]. Among the different molecular markers, SRAP and ISSR have been broadly used to evaluate species genetic diversity [
32,
33] due to their cost-effectiveness, simplicity, and versatility without requiring sequencing information [
34] and the requirement of minimal starting DNA templates [
35]. The PCR-based ISSR marker is an attractive strategy for anchoring SSR by using a single primer to amplify DNA fragments between identical microsatellite repeat regions in both directions [
35,
36]. They provide high genome coverage, high effectiveness, time effectiveness, and cost-effectiveness. The analysis of genetic diversity using ISSR markers has been successfully evaluated in several species, such as
Lolium [
37,
38,
39],
Paspalum rawitscheri (Parodi) Chase ex G.H.Rua and Valls [
40], and
Cenchrus ciliaris L. [
41]. SRAP is a PCR-based dominant marker and can amplify open reading frames (ORFs) [
42], as well as being a simple, reliable, moderate throughput ratio, and it reveals co-dominant markers [
43] and has proven to be more informative for detecting genetic diversity than other PCR-based markers [
44]. Recently, SRAP has been successfully used to study genetic variability patterns in plenty of grasses such as
Elymus breviaristatus Keng ex Keng f. [
20],
Stenotaphrum secundatum (Walter) Kuntze [
45],
Buchloe dactyloides (Nutt.) Engelm. [
46],
Dactylis glomerata L. [
47], and
Cynodon dactylon (L.) Pers. [
48].
Although RAPD markers have been used to investigate the genetic diversity of
A. lagopoides populations [
49], there is no report on the application of ISSR or SRAP markers in its genetic diversity assessment. Since
A. lagopoides is an economical, multipurpose halophyte, studies about its genetic diversity can prove helpful for its conservation strategies and breeding programs. Although several reports are available on the ecological and physiological aspects of
A. lagopoides [
50], the genetic diversity of
A. lagopoides populations from different regions of Saudi Arabia using molecular markers has not been explored so far. The present study aims to analyze the genetic diversity among/within the population of
A. lagopoides growing in different eco-geographical regions of Saudi Arabia using ISSR and SRAP markers.
4. Discussion
In conservation genetics, genetic variability is the primary study content. It is also an outcome of a long period of development, adaptation, and biological evolution. The genetic diversity of plants varies due to their evolutionary history and ecological geography [
59] and is a prerequisite for the conservation of genetic resources [
60]. Various factors such as seed dispersal, successional stages, geographical distribution range, adult density, mating system, colonization history, and natural selection can influence genetic variability within and among the plant populations [
61]. The use of molecular marker technology is an effective method for studying genetic diversity. Genetic variability of
A. lagopiodes has been conducted using RAPD [
49]. Despite being simple and convenient, RAPD marker usage is restricted due to its low stability and reproducibility. Therefore, in this work, ISSR and SRAP markers were used to study the genetic variability of
A. lagopoides collected from different eco-geographical regions of Saudi Arabia.
DNA analysis using ISSR and SRAP markers has proven to be an efficient and inexpensive way to provide molecular data for genetic diversity assessment [
42,
62]. To our knowledge, this is the first comprehensive study to evaluate the genetic diversity of
A. lagopoides populations based on inter-simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) markers.
In this study, 14 ISSR and 15 SRAP primers were employed for genetic variability among populations of A. lagopoides samples collected from five different eco-geographical regions of Saudi Arabia (Jouf, Jizan, Salwa, Qareenah, and Qaseem region). The implemented primer pairs have produced clear, visible, and optimal bands for obtaining the binary matrix, as well as the genetic diversity parameters and population genetic structure. Our results showed that both the ISSR and SRAP markers effectively revealed the genetic variability among A. lagopoides populations. However, the parameters of genetic variability such as the polymorphic bands (A = 156), average polymorphism information content (PIC = 0.34), average Nei’s gene diversity (ne = 1.330), and Shannon’s information index (I = 0.281) based on ISSR markers have slightly higher values than SRAP markers (A = 109, PIC = 0.31 ne = 1.289, and I = 0.247) in different A. lagopoides populations.
The genetic diversity revealed by the combination of SRAP and ISSR markers was highly consistent in earlier studies [
63,
64,
65]. Similar results were also reported in
Amomum tsao-ko Crevost and Lemarie [
66],
Galega officinalis L. [
60],
Auricularia auricula [
67], and
Salvia miltiorrhiza Bge [
68]. A possible explanation for these slight differences was that the ISSR markers tend to be scattered throughout the genome, which revealed the diversity of the entire genome [
35], while the SRAP markers amplified functional regions of the open reading frame [
42].
As in our findings, ISSR markers were more informative than the SRAP markers in the same way as in the genetic diversity assessment of the
Salvia miltiorrhiza Bunge [
68],
Pinellia Ten. Species [
69], and
Dioscorea L. species [
70] when using these two types of markers. However, in several other studies, SRAP proved to be more informative in assessing the genetic diversity of the
Magnolia wufengensis Pamp. [
71],
Helianthus tuberosus L. [
72], and
Ocimum species [
71] than the ISSR markers. These differences may be due to the fact that these two marker techniques target different portions of the genome, i.e., ISSR markers scattered throughout the genome, thus revealing the entire genome diversity, while SRAP markers amplified the target regions of the open reading frames(ORF), the functional groups only [
42]. These variations may also be due to marker sampling errors or polymorphism levels, emphasizing the significance of the loci count and genome coverage for accurately estimating genetic relationships among the populations [
73]. Dendrograms based on a UPGMA analysis using ISSR and SRAP data gave similar results. The Mantel test correlation revealed a strong correlation between genetic distance and geographical distance between combined analyses (r = 0.69), similar to ISSR analyses (0.73) and slightly lower than SRAP (0.80), indicating highly reliable results from the combined analysis. Similar results were reported in a study of
Codonopsis tangshen (Oliv.) D.Y. Hong [
74]. Our findings revealed that both SRAP and ISSR markers were effective and reliable, as well as accurately assessed, and revealed the genetic variability of
A. lagopoides.
Aeluropus lagopoides is predominantly a rhizomatous perennial grass that produces numerous seeds but maintains a transient seed bank. Vegetative propagation has been considered a short-term survival strategy. For a long-term strategy to maintain fitness, numerous viable seeds may, from time to time, colonize the nearest open spaces (formed due to human disturbance of frequent cutting and grazing) during years of higher-than-average precipitation [
12]. The results of molecular variance showed the genetic variability of
A. lagopoides was higher (>50) within the studied populations but varied with regions (Qaseem and Salwa 55% and Jouf 38%). A similar higher genetic diversity was also reported within
Phragmites australis (Cav.) Steud. [
75] and
Aeluropus littoralis (Gouan) Parl. Populations [
31], which also propagates vegetatively in marshy conditions. Among the five eco-geographical regions, Salwa and Qaseem populations had higher genetic diversity based on the ISSR marker analysis. For Salwa, PPB = 56% and ne = 1.380, followed by Qaseem, with PPB = 51% and ne = 1.361. At the same time, SRAP markers showed lower values.
A higher genetic diversity was found in the Qaseem region (PPB = 51% and ne = 1.379), followed by Salwa (PPB = 42% and ne = 1.261). The maximum genetic diversity found in the Qaseem region may be due to high soil fertility due to diverse vegetation, profuse seed production of A. lagopoides, out-crossing through wind pollination, and frequent rainfall, all of which set the conditions for seed germination and establishment.
Based on our field research, it could be confirmed that individuals within populations of the Qaseem region have different morphological traits because
A. lagopoides grows in a variety of distinct patches and has spikes with a good number of seeds. Similar results of higher levels of genetic diversity within the population were reported in the study of
Hertia cheirifolia (L.) [
76],
Akebia trifoliata [
77], and
Glycine tabacina [
78].
The low genetic variation in Jouf region may be due to soil nutrient deficiency and soil erosion, which are not conducive to seed germination/establishment. Populations in the Jouf region may be less diverse because they are geographically or ecologically isolated from those in the Central and Eastern regions due to the presence of several wadis, such as Wadi Arar, Wadi Aba al-Qur, etc., that pass through limestone hills and could be linked to low gene flow. The AMOVA for both ISSR and SRAP revealed that the genetic variation within the population was 60%, indicating higher genetic variability within the region, while 40% genetic variation among the population indicates a weak population structure along the regions.
The dendrograms based on Jaccard’s similarity coefficient were constructed on the ISSR and SRAP datasets to analyze genetic relationships among different
A. lagopoides populations, which were similar despite minor inconsistencies in the clustering of some subgroups. These results demonstrated that the 25
A. lagopoides populations were positively correlated with their geographical distribution. Most of the
A. lagopoides populations originating from similar regions, such as those from Jouf, were clustered in the same group and had a high level of similarity. However, the populations from Qaseem and Qareenah regions were clustered in the same group. This can be explained by the short geographic distance between the two regions (
Tables S2 and S3).
However, one of the limitations of using non-specific primers (which also includes ISSR and SRAP) and generating multilocus band patterns is the inherent nature of the studied loci being biallelic (i.e., a band is present or absent for a particular allele). Consequently, efforts to differentiate between heterozygotes (individuals with two different alleles) and homozygotes (individuals with two identical alleles) based on band intensity have not proven feasible. Thus, DNA bands are dominantly inherited markers. For the five populations studied, the obtained values of the genetic diversity parameters may have different results if the study is repeated because these parameters are sensitive to the number of individuals from each population as well as to the number of marker combinations used. The percentage of polymorphic loci may vary as more individuals are considered or more marker combinations are used, and the error value will be lower. Further research is needed to explore the standard population genetics models to evaluate the among- and within-population diversity by utilizing more marker combinations or advanced-marker molecular markers like SSR SNIPs and validate our findings.