**1. Introduction**

Salinity is a significant environmental problem that limits plant productivity, especially in arid and semiarid regions that cover approximately 40% of the globe. Semiarid regions are projected to become drier and more saline due to rising global temperatures [1–3]. Vegetation survival and productivity are primarily regulated by the water balance in soil, which affects the water balance and photosynthetic rate in plants [4]. Soils in drylands are usually heterogeneous in space and time due to the presence of biotic and abiotic elements. Spatiotemporal variations in soil salinity and water content are well documented [5]. Salinity amplifies the effects of soil drought on plants by creating additional osmotic pressure. Soil is considered saline when the salt content exceeds 3–5 g salt/L in the soil solution, when electrical conductivity (EC) exceeds 2–4 dS/m, or when the sum of salts exceeds 0.15–0.2%, creating osmotic pressure above 0.2 MPa, which significantly reduces the yield of the most crops [2,6]. Salinity reduces plant growth and prematurely ages mature leaves, which leads to a decrease in the functional leaf area. A decrease in plant biomass is also influenced by Na<sup>+</sup> and Cl<sup>−</sup> toxicity and the accompanying oxidative stress [2,7]. Halophytes are highly salt-tolerant plants but underutilized resources that occupy naturally saline soil environments in coastal estuaries and inland salt flats in arid and semiarid zones [8]. Nowadays, climate-smart agriculture (CSA) practices increasingly use wild

**Citation:** Shuyskaya, E.; Toderich, K.; Kolesnikov, A.; Prokofieva, M.; Lebedeva, M. Effects of Vertically Heterogeneous Soil Salinity on Genetic Polymorphism and Productivity of the Widespread Halophyte *Bassia prostrata*. *Life* **2023**, *13*, 56. https://doi.org/10.3390/ life13010056

Academic Editors: Hakim Manghwar and Wajid Zaman

Received: 23 November 2022 Revised: 17 December 2022 Accepted: 21 December 2022 Published: 24 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

salt-tolerant species (halophytes) to restore the grazing capacity of degraded pastures, provide forage for livestock and utilize oilseeds and medicinal and aromatic plants [7,9]. Apart from these applications, halophytes play a significant role in the maintenance of ecosystem functions and sustainability [10,11].

Genetic diversity provides plants with the ability to adapt and survive in changing environments, including soil chemistry variability. The genetic architecture of a population plays a fundamental role in the origin and maintenance of local adaptation [12]. The degree of local adaptation is largely determined by the interaction between selection and gene flow along ecological gradients. Different types of selection can operate under natural conditions: (i) conditionally neutral selection occurs when two alleles do not have an advantage in fitness in one environment but differ in fitness in another environment; (ii) environmentally antagonistic selection, when different alleles are locally adapted to different environments, conferring higher fitness there [12]. 'Fitness' is often viewed as the ability to withstand adverse conditions; however, from an evolutionary perspective, fitness is defined as the ability of an individual to spread their genes through offspring. Thus, in plants, fitness depends on the number of seeds that a plant can successfully produce under adverse environmental conditions [13]. A common plant response to environmental stresses is a decrease in fertility, which consists of aborting ovules and/or pollen and redirecting resources from reproductive activity into metabolic reactions for stress tolerance [14]. Plants are reported to control the consumption of maternal resources at several stages of development by regulating the number of flowers, gametophytes, and embryos that develop further [14]. This type of developmental regulation can lead to the favored selection of certain alleles or genotypes, producing genotype–environment associations and/or interactions [15]. The survival rate and adaptation of populations in different and changing environments depend on the genetic diversity of the seed pool. For example, diversity in the genetic composition of seeds allows *Atriplex tatarica* to survive under distinct conditions: heterozygous plants mainly germinate under optimal conditions, and homozygous plants typically germinate under suboptimal conditions [16]. Ecological factors that influence reproduction and seed dispersal are, therefore, particularly important aspects in shaping genetic diversity and population structures. Edaphic conditions, such as soil type, pH, nutrients, moisture, and the depth of soil layers, can significantly affect the level of genetic diversity and local adaptation in plant populations [15,17–19]. For example, *Phragmites australis* populations with high genetic diversity have a high tolerance to soil salinity [20]. The high genetic diversity of populations is fundamental to the long-term survival success of a plant species [21].

In unfavorable environments, such as areas of high soil salinity, plants are forced to seek a 'compromise' between productivity and adaptation, which depends on genetic diversity at the population level. Within the soil profile, salinity is a dynamic indicator; it changes with depth and according to seasons [5]. In turn, plants have a vertical fine-root distribution, which determines the possibility of acquiring resources along the soil profile, since plants rely mainly on their fine roots to acquire belowground water and nutrient resources [22]. To assess and predict the productivity and adaptation of species under changing conditions, it is necessary to understand how the salinization of different soil layers affects biomass and genetic polymorphism formation. A convenient model species for these purposes is the polymorphic widespread halophyte *Bassia prostrata* (L.) A.J. Scott (*Kochia prostrata* (L.) Schrad.) (subfamily Chenopodiaceae), with a significant variety of morphological, biochemical, and ecological–physiological properties; high genetic polymorphism; and wide ecological plasticity [23–27]. Moreover, the effect of soil conditions on both the level of diversity and genetic structure of *B. prostrata* populations has been shown [28].

The present study aims to investigate the effects of the level and chemistry of salinity within different soil layers (including horizontal and vertical variations in the soil characteristics) on the productivity and genetic diversity of the halophyte *B. prostrata* to clarify

the adaptive mechanism it uses to withstand fluctuations of salt accumulation along soil depth profiles.

#### **2. Materials and Methods**

#### *2.1. Study Area*

Studies were carried out in the northwestern Caspian Lowland (Russia) (Figure 1A). The region is a flat marine accumulative plain, which is characterized by the almost complete absence of surface and subsurface runoff. According to climate parameters (Figure 1B), the region is arid, with an average annual temperature of 8.7 ◦C and precipitation of 291 mm. In Caspian Lowland plain landscapes, solonetzic complexes are widespread; depressions and other negative relief elements (microdepressions, depressions, estuaries) are characterized by dark-colored chernozem-like or meadow–chestnut soils [29]. Seven typical habitats of *B. prostrata* were selected for the study based on differences in the soil salinity levels (No. 1–7 in Figure 1A). Five of them were located near the salt lakes Bulukhta and Elton at different distances from the coastline. The other two habitats were in the plain part between these lakes (Figure 1A). *B. prostrata* habitats were characterized mainly by solonetzic and/or light chestnut solonetzic soils and desert steppe vegetation.

**Figure 1.** The location of seven populations of *Bassia prostrata* (**A**) and the long-term (2007–2018) average atmospheric temperature and precipitation (**B**) of the northwestern Caspian Lowland. 1–7—numbers of populations (habitats); Tm—temperature; P—precipitation.

#### *2.2. Plant Sampling*

*Bassia prostrata* (L.) A.J. Scott (*Kochia prostrata* (L.) Schrad.) (Chenopodiaceae) is a typical perennial C4 halophyte native to arid and semiarid rangelands in Central Eurasia and the Western United States. *B. prostrata* naturally occurs in all kinds of soils, such as saline, sandy, rocky, and poor soils [24,30,31]. *B. prostrata* has a thick, woody root system that can penetrate 3–6.5 m depths and lateral roots stretching 130–160 cm that mine for moisture in the upper (up to 60 cm) soil layers [30,31]. This is the reason for studying

the upper soil layers: approximately 0–20 cm, 20–40 cm, and 40–60 cm. Soils, plants, and seeds were sampled in seven typical habitats of *B. prostrata* (No. 1–7 in Figure 1A). The aboveground parts of five plants were harvested in each habitat in the middle of September for biomass analysis. More than 100 seeds from 10 to 15 mother plants from each habitat (population) were collected at the beginning of November and combined to generate a seed pool for population genetic analysis.

#### *2.3. Soil Sampling and Analysis*

Seven habitats of *B. prostrata* soil pits (Nos. 14, 11, 15, 10, 18, 7, and 6, corresponding to habitats Nos. 1 to 7 in Figure 1A) were excavated. Profiles were examined to depths of 0 to 60 cm. Three soil samples (*n* = 3) were used for the analysis of each soil layer of each habitat. Chemical and physicochemical analyses were performed at the Analytical Laboratory of the V.V. Dokuchaev Soil Science Institute using standard methods [32]. Calcium and magnesium concentrations in water extracts (1:5) were determined with the complexometric titration method; sodium and potassium concentrations were determined with the flame photometry method; the total alkalinity was determined using titration with sulfuric acid (with methyl orange indicator); the concentration of chlorine ions was determined with argentometry (according to Mohr); and the concentration of sulfate ions was determined using titration with BaCl2. The content of ions Na+, K+, Ca2+, Mg2+, Cl−, SO4 <sup>2</sup>−, and HCO3 <sup>−</sup> are presented in cmol(eq)/kg. The sum of salts (Ssalts) represents the sum of the mass fraction of ions from the solid soil residue (%) [6].

#### *2.4. Plant Biomass and Water Content*

Plant height, fresh (FW) and dry (DW) biomass, and water content (W) were assessed for aboveground parts of *B. prostrata* plants (*n* = 5) from seven habitats. Biomass was estimated for fresh and dry shoots. Plant samples were dried at 80 ◦C for two days until reaching a constant mass to quantitatively measure the dry shoot matter. The water content in the shoots was calculated according to the following formula:

$$\mathbf{W} = (\mathbf{F}\mathbf{W} - \mathbf{D}\mathbf{W}) / \mathbf{D}\mathbf{W}.\tag{1}$$

#### *2.5. Population Genetic Analysis*

Genetic diversity can be studied using neutral markers (based on differences in DNA sequences) and partially selective markers (isozymes), which can reflect changes in environmental conditions [33,34]. In this study, we used isozymes (alternative forms of the enzymes encoded by different alleles of the same gene) to assess the genetic diversity of the populations.

For each population of *B. prostrata*, 50 seeds from the seed pool (more than 100 seeds from 10 to 15 mother plants) were germinated, and all good germinated seeds (*n* = 25–35 per population) were analyzed for genetic polymorphism. Population genetic analysis was performed on embryos using starch gel electrophoresis of the following enzymatic systems: glutamate oxaloacetate transaminase (GOT (AAT), E.C. 2.6.1.1), diaphorase (DIA, E.C. 1.6.99), glutamate dehydrogenase (GDH, E.C. 1.4.1.2), superoxide dismutase (SOD, E.C. 1.15.1.1), glucose-6-phosphate dehydrogenase (G6PD, E.C. 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGD, E.C. 1.1.1.44), malate dehydrogenase (MDH, E.C. 1.1.1.37), and malic enzyme (Me, E.C. 1.1.1.40). The seeds were cleaned of their wings and soaked in water for 12 h and homogenized in 80 μL of Tris-HCl buffer with KCl, MgCl2, EDTA, Triton X-100, and PVP. Enzymes were separated in 10% starch gel using two buffer systems. In system 1, the electrode buffer was 160 mM Tris–50 mM citric acid, pH 8.0; the gel buffer was prepared by diluting 10 mL of the electrode buffer with 90 mL H2O. In system 2, the electrode buffer was 300 mM boric acid–60 mM NaOH, pH 8.2; the gel buffer was 80 mM Tris–9 mM citric acid, pH 8.7. Electrophoresis was performed at 90 V, 40–50 mA in buffer system 1 or at 210 V, and 70–80 mA in buffer system 2 for 4–6 h at 5 ◦C. Staining of particular enzymes and genetic interpretation of the results followed standard techniques according to Soltis and Soltis [35] and Spooner et al. [33]. The level of genetic polymorphism was estimated

by calculating observed (*H*o) and expected (*H*e) heterozygosity for each polymorphic loci and by calculating the proportion of polymorphic loci (P99) and the average (for all loci) observed (*H*o) and expected (*H*e) heterozygosity in POPGEN 1.32.

#### *2.6. Statistical Analysis*

Principal component analysis (PCA) was carried out using R software (version 3.6.1). Table 1 and Figure 2 show the means of the obtained values and their standard errors (*n* = 3 for soil samples and *n* = 5 for plant samples).
