Assessment of Genetic Variation in Natural Populations of Hippophae rhamnoides L. from Kazakhstan Using Retrotransposon-Based Markers

Abstract

Analysis of the genetic diversity of natural populations of economically valuable plants is important for conservation and selection strategies. In this study, the genetic diversity of 11 natural populations of Hippophae rhamnoides L.—sea buckthorn from different regions of Kazakhstan—was studied using Inter-Primer Binding Site Polymorphism (iPBS) markers based on conserved sequences of tRNA primer-binding sites (PBSs) that initiate retrotransposon replication. Universal PBS primers yielded reproducible and informative amplicons, forming unique profiles for each sample. Analysis of molecular variance showed that 60% of the total genetic variation was due to intrapopulation differences and 40% was due to interpopulation differentiation. The highest genetic diversity was found in the Shetlasty and Tersayryk sea buckthorn populations, whereas the Karatal and Topkain populations were characterised by minimal values, although unique alleles were observed in the latter population, indicating possible adaptation to local environmental conditions or genetic isolation. Principal coordinate analysis, UPGMA clustering, and Bayesian structure analysis (K = 4) confirmed geographical structuring. This study provides insights into the genetic structure of sea buckthorn populations in Kazakhstan and demonstrates the effectiveness of iPBS markers for assessing intraspecific diversity. The obtained results provide a basis for the conversation of H. rhamnoides gene pool and underscore the need for both in situ conservation of genetically rich populations and ex situ protection of vulnerable groups.

1. Introduction

According to the latest classification of Plants of the World Online, the small genus Hippophae of the family Elaeagnaceae includes seven species of considerable ecological, nutritional, and medicinal value [1,2]. The most widely distributed species is Hippophae rhamnoides L. (Elaeagnaceae Juss.), a thorny shrub with an extensive range, covering many regions of Europe, Asia, and North America [3,4]. H. rhamnoides is a polymorphic species with considerable ecological adaptability, which influences its phenotypic and genotypic variability [3,5,6,7]. In Kazakhstan, natural populations of the species occupy approximately 2640 ha. The species is distributed in the Eastern Uplands, Zaisan, Pribalkhashye, Altai and Tarbagatai, Dzungarian, Zailiysky, Kyrgyz, Kungey and Terskey Alatau, Ketmen Ridge, Western Tien Shan, Karatau and other upland and mountainous regions [8,9,10]. The habitats of natural populations in Kazakhstan are confined to the well-drained soils of river channels with a light mechanical composition, where the flowing groundwater is shallow [8].

Sea buckthorn has been used since ancient times as a fruit, vitamin, medicinal, and ornamental plant. This plant has long been used as a medicinal and food supplement [11,12,13,14]. Historical records of ancient Chinese, Indian, and Tibetan medicine, ancient Greek texts, and Ayurveda and species are included in the Chinese Pharmacopoeia [4,15,16,17,18,19]. Extracts of H. rhamnoides L. are beneficial for health and exhibit anti-inflammatory, antioxidant, hepatoprotective, anticancer, hypoglycaemic, hypolipidaemic, and antibacterial properties [17,20,21,22,23,24]. Sea buckthorn fruits and other plant organs contain more than 200 types of nutritional (proteins, minerals) and bioactive compounds have been identified in sea buckthorn, such as polyunsaturated fatty acids, carotenoids, flavonoids, sugar alcohols, lignans, essential oils, tannins, terpenoids, organic acids, superoxide dismutase, and phytosterols, and vitamins C and E which, supporting immune function, cardiovascular health, and protection against oxidative stress [13,17,20,21,22,23,24,25,26,27,28,29,30,31,32].

Research on the introduction of sea buckthorn from wild populations began in the mid-19th century; selection and breeding efforts have resulted in more than 150 varieties for culinary and/or medical use [33].

Since 1981, research has been conducted at the Altai Botanical Garden to preserve the valuable gene pool of this species. Research on the introduction and selection of wild sea buckthorn identified genetic donors with economically valuable traits (ecological and economic adaptability, fruit size, taste quality, and nutritional value), and potential forms and seedlings were selected for further selection. After many years of selection, several varieties have been created and protected by patents from the Republic of Kazakhstan [34]. These studies indicate the high genetic diversity of the H. rhamnoides gene pool from Kazakh Altai, establishing it as an extremely valuable resource for the conservation of biological diversity and a promising source material for breeding programs.

The study of the genetic diversity of Kazakhstani populations of H. rhamnoides was conducted using classical botanical methods [8,35,36,37]. Molecular markers are widely used as effective tools for analysing the genetic diversity of H. rhamnoides, including establishing the taxonomic and geographical origins of varieties, determining the sex of plants, and studying the population genetic structure. Studies on sea buckthorn (H. rhamnoides) enable more accurate detection of intraspecific differentiation, tracking evolutionary relationships, and selecting promising forms for breeding programs [38].

Early studies on genetic diversity in sea buckthorn used isozyme markers and Random Amplified Polymorphic DNA (RAPD), which revealed limited interpopulation differentiation but considerable intra- and interspecific variability [39,40,41,42,43].

To assess genetic differentiation and analyse intraspecific and interspecific variability in various taxa of the genus Hippophae, various marker systems were used, such as simple sequence repeat (SSR) markers, amplified fragment length polymorphism (AFLP), and selective amplification of microsatellite polymorphic loci (SAMPL) [38,40,44,45,46,47,48]. Using expressed sequence tags-simple sequence repeats (EST-SSRs), significant differentiation was observed between sea buckthorn populations as well as between wild and agricultural plantations [44,45]. Inter-simple sequence repeat (ISSR) markers have also been widely used due to their simplicity, reproducibility, and high discriminatory power, and they have even facilitated the development of sex-specific markers for H. rhamnoides spp. turkestanica and related taxa [49,50,51,52,53,54,55]. Despite these advances, questions remain regarding the extent of genetic diversity in natural populations and its relevance for breeding programs. Clarifying this variability is essential for conservation and the effective utilization of sea buckthorn genetic resources.

To study intraspecific genetic polymorphisms among various species of the genus Hippophae, it is appropriate to use markers characterised by a wide distribution in the genome and, importantly, that are universal for all species, including those that have not been investigated. Currently, among the various classes of molecular markers used to analyse the genetic polymorphisms of H. rhamnoides L., universal markers based on retrotransposons have never been used.

Almost all eukaryotic organisms are characterised by a high content of tetratransposons in their genome, including LTR and non-LTR retrotransposons [56,57]. Mobile genetic elements that move during the replication of the LTR retrotransposon via the reverse transcription of RNA and integration of the resulting cDNA into another locus develop unique combinations of highly conserved sequences. These sequences, including the tRNA primer-binding site (PBS), can be identified by PCR. In this case, the sizes of the amplified fragments depend on the frequency of repeats in the studied genome; the less frequently the iPBS sequence occurs in the genome, the larger the amplicon size, and vice versa [58].

This universal genomic fingerprinting system has been successfully used for the rapid detection of molecular genetic polymorphisms in eukaryotic organisms, including Vitis sp., Phaseolus vulgaris L., Castanea sativa Mill., Paeonia anomala, phytopathogenic fungi, and other organisms [2,59,60,61,62].

This approach provides universal applicability across all eukaryotic organisms, technical simplicity, and cost-effectiveness, as well as enables the identification of individual genetic polymorphisms, even in closely related genotypes, owing to the length of the primers and the high annealing temperature [63].

Notably, retrotransposons represent a reservoir of potential genomic instability that determines the evolutionary diversity and genetic plasticity of species and their functional variability [60]. Insertions of mobile elements induced by various stress factors, such as pathogens, drought, increased insolation, and extreme temperatures, are accompanied by genetic rearrangements [64,65,66]. These processes occur predominantly in regions of active euchromatin and lead to restructuring of the body’s regulatory systems, establishing survival strategies that can be fixed at the genetic level [67,68]. This is pronounced in plants with vegetative reproduction, where somatic changes can be transmitted to the offspring [69,70,71].

The high adaptability of sea buckthorn to various environmental conditions results in the emergence of multiple ecotypes that differ in morphometric characteristics as well as genetically [72,73]. Intraspecific divergence is a key factor for survival and reproductive success, ensuring the ability of populations to adapt to unfavourable environmental conditions, increase their habitats, and effectively resist selective pressure from natural selection [74]. Therefore, the study of intraspecific genetic variability is particularly relevant because it facilitates the identification of adaptation mechanisms, predicts evolutionary trajectories, and develops strategies to preserve biodiversity. The novelty of this study lies in the first application of iPBS retrotransposon markers to assess the genetic diversity of wild H. rhamnoides populations in Kazakhstan.

In this study, we investigated the genetic diversity of sea buckthorn, using samples from wild populations in Kazakhstan and potential samples isolated as a result of analytical selection from introduced plants.

2. Materials and Methods

2.1. Plant Material and DNA Extraction

Sea buckthorn (Hippophae rhamnoides L.) was collected from various regions of Kazakhstan in the period 2023–2025, covering a wide range of ecological and geographical growth conditions of this species (Figure 1). Herbarium samples from the Altai Botanical Garden, collected during expeditions from 1981 to 1984, were also used in this study. All samples were classified into three main categories: wild sea buckthorn, varieties and selection forms, and herbarium samples.

Consequently, a representative collection was established to evaluate the intraspecific variability and potential value of the studied forms for the selection and conservation of the H. rhamnoides gene pool in Kazakhstan.

Each sample was provided with a unique ID number, and the coordinates (latitude and longitude) and sample type for each exact collection location are presented in

Table 1. Characteristics of the habitat conditions of H. rhamnoides populations in the Kazakhstan Altai region.

Populations	Location	Sample Type	Sample Accession	Original Number
Kendyrlyk	47.47830 85.21473	Varieties	Feyerverk	K-14-82 (3−24) III
			Fakel	K-14-81(3−25) III
			Krasnoplodnaja	K-14-81(4−27)
		Wild population	Ken-14-86	K-14-86(4−18)
			Ken-4-86	K-4-86(4−28)
			Ken-14-81	K-14-81(3−38)
		Herbarium	Ken-1-HRB	Kendyrlyk-84−1
			Ken-6-HRB	Kendyrlyk-84−6
			Ken-2-HRB	Kendyrlyk-84−2
			Ken-3-HRB	Kendyrlyk-8−-3
Shetlasty	47.16724 84.09574	Varieties	Asem	Sh-9-81 (3−27)
			Shetlastinka	Sh-7 (2−24) III
			Jantarnaja	Sh-12 05 (2−1) III
			Solnyshko	Sh-12-81 (1−18)
			Yozhik	Sh-3−84
		Wild population	Sh-4-18	Sh-9-81(4−18) IV
			Sh-4-20	Sh-9-81(4−20) IV
			Sh-4-15	Sh-9-81(4−15)
			Sh-5-27	Sh-20-84(5−27)
			Sh-5-11	Sh-16-86(5−11)
			Sh-4-5	Sh-9-81(4−5)
		Herbarium	Sh-1-HRB	Shetlasty-4−84
			Sh-2-HRB	Shetlasty-3−84
			Sh-3-HRB	Shetlasty-7−84
			Sh-4-HRB	Shetlasty-13−84
			Sh-5-HRB	Shetlasty-9−84
			Sh-6-HRB	Shetlasty-2 −84
Kaindysu	47.585513 83.64861	Wild population	Kan-3-34 IV	Kan-2-84 (3−34) IV
			Kan-4-6	Kan-9-8 (4−6) IV
			Kan-1-5	Kan-3-82 (1−5)
			Kan-3-1	Kan-3-82 (3−1)
			Kan-8-4	Kan-8-81 (8−4)
			Kan-3-34	Kan-2-84 (3−34)
Tersayryk	48.16005 85.16727	Varieties	Plakuchaja	T-14-82 (2−32)
			Jubilejnaja Kotuhova	T-2-82 (2−22) III
		Wild population	T-3-26	T-14-86 (3−26)
			T-2-32	T-2-82 (2−32)
			T-6-29	T-14-82 (6−29)
			T-4-3	T-3-83 (4−3)
			T-4-4	T-3-83 (4−4)
			T-3-20	T-14-86 (3−20)
			T-4-16	T-17-82 (4−16)
			T-2-32	T-14-82 (2−32)
			T-4-32	T-7-86 (4−32)
		Herbarium	T-3-HRB	Tersayryk 3-86
			T-7-HRB	Tersayryk 7-86
			T-1-HRB	Tersayryk 1-86
			T-5-HRB	Tersayryk 5-86
Karatal	47.62766 85.20537	Wild population	Ks-1-24	Karatal KP 1-24
		Herbarium	Ks-1-HRB	Karatal K-3
			Ks-2-HRB	Karatal KP
Topkain	49.17389 85.51889	Herbarium	Top-1-HRB	Topkain 3-84
			Top-2-HRB	Topkain 5-84
Manyrak	47.39027 83.87944	Wild population	Man-3-24	Manyrak 3-24
			Man-12-24	Manyrak 12-24
			Man-7-24	Manyrak 7-24
Almaty	43.32902 78.54828	Wild population	ALM-1	Almaty-24-1
	43.132895 76.545421		ALM-2	Almaty-24-2
	43.064783 76.544676		ALM-3	Almaty-24-3
	43.043301 76.542341		ALM-4	Almaty-24-4
	43.02056 78.14644		ALM-5	Almaty-24-5
Akmola	52.912467 72.912467	Wild population	AKML-1	Akmola-24-1
	52.912467 72.922467		AKML-2	Akmola-24-2
	51.074516 71.765893	Wild population	AST-1	Astana-24-11
	52.46106 70.34146	Herbarium	AKML-1-HRB	AKML-17-1
Kostanay	51.33319 64.13590	Herbarium	Kos-1-HRB	Kostanay-23-1
		Herbarium	Kos-2-HRB	Kostanay-23-2
Karaganda	49.753249 73.043323	Herbarium	Kar-1-HRB	Karaganda-23-1
		Herbarium	Kar-2-HRB	Karaganda-23-2

Note: All sea buckthorn cultivars were developed by Dr. T.A. Vdovina through analytical selection over three generations from introduced accessions of Hippophae rhamnoides. For the samples from Astana, Kostanay, and Karaganda, herbarium specimens from the collection of the National Center for Biotechnology (Astana, Kazakhstan) were used, as well as samples from wild-growing thickets in the vicinity of Astana. The herbarium specimens from East Kazakhstan Region were collected during expedition studies conducted in 1984–1986.

.

The herbarium specimens were designed in accordance with the requirements of botanical herbarisation and provided with full geographical and taxonomic annotations. Varietal and selected samples were collected mainly from the experimental plots of the Altai Botanical Garden. Herbarium materials and wild forms originated from various natural landscapes, including gorges, mountain slopes, riverbanks, and flat and forested areas. The highest mountain samples were obtained at certain heights (Almaty region, Kungey Alatau ridge), which provided material for a comparative analysis of adaptation mechanisms depending on the altitude zone.

A distribution map of H. rhamnoides in Kazakhstan was developed using Quantum Geographic Information System (QGIS) (Figure 2). The regional base layer was digitised from official cartographic sources, and five floristic zones were delineated: Kostanay, Akmola, Karaganda, Almaty, and East Kazakhstan regions. Sampling sites were georeferenced and plotted as red points, while the total number of specimens collected in each region was aggregated and visualised by teal circles with numbers indicating sample counts.

The results show a pronounced concentration of samples in the East Kazakhstan region (56 samples), whereas other areas such as Akmola and Almaty contained smaller yet relevant collections (2–5 samples). Specific collection sites, including Topkain (2), Kaindysu (6), Tersayryk (15), Karatal (3), Kendyryk (10), Shetlasty (17), and Manyrak (3), are labelled to highlight local hotspots. This spatial distribution reflects both the ecological prevalence of H. rhamnoides populations and the targeted efforts of field expeditions and herbarium surveys in the country.

2.2. DNA Extraction and iPBS Genotyping

The plant material was placed in Zipper bags (Minigrip^®, Seguin, TX, USA), stored on ice during field transportation, and preserved at −20 °C in the laboratory. DNA extraction was performed on individual H. rhamnoides L. leaves using a high-salt gel electroelution trap or an acidic CTAB solution (2% CTAB, 2 M NaCl, 10 mM Na₃EDTA, and 50 mM HEPES, pH 5.3) [75,76,77]. DNA detection was performed by electrophoresis on a 1% agarose gel placed in a chamber with 1 × THE buffer (20 mM Tris-HEPES, pH 8.06), and gel scanning was conducted using the iBright™ CL1500 Imaging System (Invitrogen™, Waltham, MA, USA) gel documentation system.

2.3. Genetic Diversity Assessment

Universal PBS primers were used to evaluate the genetic diversity of the H. rhamnoides population. The sequences of the primers complementary to various retrotransposon regions are listed in

Table 2. Characteristics of PBS primers used for H. rhamnoides genetic diversity analysis.

ID	Sequence	Tm	GC (%)	LC (%)
2225	AGCATAGCTTTGATACCA	54.1	38.9	80
2232	AGAGAGGCTCGGATACCA	60.9	55.6	83
2237	CCCCTACCTGGCGTGCCA	69.6	72.2	77
2239	ACCTAGGCTCGGATGCCA	65.0	61.1	91
2240	AACCTGGCTCAGATGCCA	63.2	55.6	88
2247	AACCTGGCTCTGATACCA	55.9	50	83
2252	TCATGGCTCATGATACCA	57.1	44.4	80
2300	CACCGGGCTCTGATACCA	64.0	61.1	90
2374	CCCAGCAAACCA	48.2	58.1	79

.

2.4. PCR Reaction and Data Analysis

The PCR reaction system (20 µL) included 10 ng DNA, 1 × Phire Reaction Buffer with MgCl₂, 1 µM PBS primer, 0.2 mM dNTP mixture, and 0.5 U Phire Hot Start II DNA Polymerase. The reaction procedure involved pre-denaturation at 98 °C for 2 min, and then 30 cycles of 98 °C for 10 s, 55–60 °C for 30 s (depending on the primer set annealing temperature), 72 °C for 1 min, and an additional elongation at 72 °C for 2 min. The amplification was performed using a SimpliAmp thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). PCR products were detected by a 1.5% agarose gel stained with ethidium bromide and photographed by a gel documentation system using the iBright™ CL1500 Imaging System (Invitrogen) at the end of electrophoresis. DNA fragment sizes were determined by comparison with a marker (Thermo Fisher Scientific GeneRuler DNA Ladder Mix; 100–10,000 bp).

2.5. Data Analysis

Only distinct and scorable amplicons were considered for genetic variability analysis. The polymorphism level specific to each primer was calculated as the ratio of the polymorphic amplicons to the total number of amplicons, assuming that each unique band size represented a unique locus. The bands in iPBS electrophoresis profiles were counted using Excel 2019 and assigned corresponding “1” or “0” values based on the presence or absence of bands, respectively. These data were used to calculate the total number of alleles, Shannon’s information index (I), genetic differentiation index (PhiPT) among populations, and the number of private alleles per population using the GenAlex 6.5 software [78]. Molecular variance analysis (AMOVA) among and within populations was performed using GenAlex 6.5.

STRUCTURE version 2.3.4 software was used to obtain the hierarchical organisation of the genetic structure of the 11 populations, and the admixture model was selected as ancestry. Bayesian-based software, that estimates the number of genetic clusters (K) and evaluates admixture levels among them, was also used. The analysis was conducted for K values ranging from 1 to 10. Each run included a burn-in period of 100,000 iterations, followed by 500,000 Markov Chain Monte Carlo (MCMC) iterations [79]. The dataset included 69 individuals genotyped at 47 loci. The number of genetic groups (K) was determined using Evanno’s method [80,81]. Graphical visualisation of population structure was generated using the CLUMPAK version 1.1 online resource [82].

3. Results

3.1. Analysis of iPBS Loci Variability in H. rhamnoides

Evaluation of genetic diversity using PBS markers demonstrated their applicability to H. rhamnoides. All 9 primers were considered satisfactory as they consistently produced more than five clear bands, including polymorphic fragments. Electrophoretic separation of the PCR products produced amplicons ranging from 100 to 5000 bp in size. All the primers were highly informative. The most informative markers were: 2252 (0.961), 2374 (0.907), 2390 (0.900), 2247 (0.982). All primers were reliable for genetic polymorphism analysis, and markers with PIC > 0.90. Unique profiles consisting of clear and assessable bands were generated for each sample. Their distribution profiles depended on the specific population and primers used (Figures S1–S9).

3.2. Genetic Differentiation of H. rhamnoides Populations Revealed by iPBS Markers

Differences in amplification patterns using PBS primers indicate the level of intraspecific polymorphisms and demonstrate the diversity of retrotransposon insertions in the genomes of representatives from different geographic groups. The data obtained confirmed the effectiveness of using iPBS markers to assess the genetic variability and population structure of the species under study. Each individual profile from a specific H. rhamnoides population was studied by genomic fingerprinting, which determined the allele frequencies of the iPBS amplicons. The indices of genetic diversity of the samples, based on the results of iPBS profiling, are presented in

Table 3. Genetic variation in H. rhamnoides populations based on iPBS markers.

Population	Na	Ne	I	He	uHe	PB, %	Number of Private Bands
Shetlasty	1.322	1.320	0.296	0.194	0.20	60.0	1
Kaindysu	0.965	1.234	0.202	0.135	0.148	37.4	0
Kendyrlyk	1.017	1.199	0.189	0.122	0.129	41.7	0
Tersayryk	1.304	1.258	0.258	0.164	0.169	59.1	5
Manyrak	0.722	1.208	0.162	0.113	0.135	26.1	0
Karatal	0.617	1.116	0.099	0.067	0.08	17.4	0
Almaty	0.783	1.189	0.158	0.108	0.12	26.9	0
Akmola	0.704	1.131	0.109	0.075	0.085	18.3	0
Kostanay	0.774	1.172	0.147	0.101	0.134	24.4	0
Karaganda	0.843	1.221	0.189	0.13	0.173	31.3	0
Topkain	0.548	1.061	0.053	0.036	0.048	8.7	2
Mean	0.873	1.192	0.169	0.113	0.129	31.9
SE	0.024	0.009	0.007	0.005	0.006	4.9

Note: Na, number of alleles per locus; Ne, effective number of alleles; I, Shannon’s information index; He, expected heterozygosity; uHe, unbiased expected heterozygosity; PB, % of polymorphic loci; SE, standard error.

.

The highest Na values were observed in the Shetlasty and Tersayryk populations, where the mean number of alleles detected per locus (Na) was 1.322 and 1.304, respectively. Slightly lower values were recorded for the Kendyrlyk (1.017) and Kaindysu (0.965) populations. The remaining populations had below-average Na values below the species average. The lowest values were observed in the Karatal (0.617) and Topkain (0.548) populations.

The effective number of alleles (Ne) reached a maximum in the Shetlasty (1.320) and Tersayryk (1.258) populations. In four populations, namely Kaindysu (1.234), Karaganda (1.221), Monyrak (1.208), and Kendyrlyk (1.199), the allele frequency distribution values exceeded the species average. The remaining populations exhibited values below the mean. Only in the Shetlasty and Tersayryk populations was Na > Ne, which may be attributed to the presence of unique rare alleles in these populations as confirmed by fingerprint analysis. Two unique alleles were also detected in the Topkain population, despite having the lowest genetic diversity values, which may indicate population isolation or accumulation of its own mutations.

As expected, the genetic diversity Shannon index (I) showed the highest values in the Shetlasty (0.296) and Tersayryk (0.258) populations, confirming a higher degree of diversity and evenness in the distribution of PBS alleles. The lowest values of the Shannon genetic diversity index I, heterozygosity (He), and iPBS locus polymorphisms were observed in the Karatal and Topkain populations.

Molecular variance analysis (AMOVA) revealed that 40% of the total genetic variation was attributable to differences among H. rhamnoides populations, whereas 60% was attributable to variations within populations (

Table 4. Molecular variance analysis (AMOVA) of H. rhamnoides based on PBS profiling.

Variability	Df	SS	MS	Est. Var.	%	PhiPT	p Value
Among populations	10	544.953	54.495	7.417	40%	0.401	0.001
Within populations	58	642.09	11.071	11.071	60%
General	68	1187.043		18.487	100%

Note: Df—Number of degrees of freedom; SS—Sum of squares; MS—Mean square; Est. Var—Variance; PhiPT—Index of population genetic differentiation; p < 0.05 considered statistically significant.

).

The total variance was 18.487, of which 7.417 was attributed to the among-population component and 11.071 to the within-population component. The estimated value of genetic differentiation, PhiPT = 0.401 (p = 0.001), indicated a high level of population structure and statistically significant differences among H. rhamnoides populations.

The pairwise genetic distance matrix revealed closely related and highly differentiated populations (

Table 5. Pairwise population matrix of Nei’s genetic distance.

Shetlasty	Kaindysu	Kendyrlyk	Tersayryk	Monyrak	Karakatal	Almaty	Akmola	Kostanay	Karaganda
0.145	0									Kaindysu
0.174	0.130	0								Kendyrlyk
0.103	0.171	0.137	0							Tersayryk
0.148	0.213	0.215	0.143	0						Monyrak
0.238	0.179	0.182	0.140	0.228	0					Karakatal
0.135	0.223	0.248	0.147	0.259	0.252	0				Almaty
0.261	0.242	0.31	0.268	0.327	0.248	0.342	0			Akmola
0.193	0.186	0.256	0.211	0.252	0.234	0.288	0.126	0		Kostanay
0.202	0.202	0.221	0.174	0.304	0.241	0.237	0.231	0.131	0	Karaganda
0.314	0.354	0.375	0.428	0.42	0.436	0.401	0.412	0.384	0.384	Topkain

). Analysis of genetic distances revealed substantial differences between the studied populations. The shortest distances were observed between the Shetlasty and Tersayryk (0.103), Shetlasty and Kendyrlyk (0.174), and Kaindysu and Kendyrlyk populations (0.130).

In contrast, the greatest distances were recorded between the Topkain population and all the other samples (0.314–0.436), confirming its pronounced genetic distinctiveness. Notably substantial differences were observed in the Karatal (0.436), Monyrak (0.420), and Shetlasty (0.314) populations.

The northern populations (Akmola, Kostanay, and Karaganda) demonstrated intermediate distance values (0.126–0.288) and formed a relatively homogeneous cluster, which was consistent with the results of principal coordinate analysis.

3.3. PCoA Analysis and Clustering for the Assessment of the Genetic Structure of H. rhamnoides Germplasm Based on iPBS Profiling

Principal coordinate analysis (PCoA) showed that the first three axes together accounted for 57.53% of the total variation (Figure 3). The first axis contributed the largest proportion, explaining 23.59% of the variability. The second axis explained 21.04%, and the third axis 12.9%. Thus, the two-dimensional representation (axes 1 and 2) captured 44.63% of the total variability, revealing the main patterns of differentiation among the Kazakhstani H. rhamnoides samples.

Samples from the Akmola, Kostanay, and Karaganda regions were clustered in the upper part of the plot, indicating their genetic similarity and geographic proximity. The Almaty, Monyrak, Kendyrlyk, Shetlasty, and Tersayryk populations formed a compact cluster in the lower left part of the diagram, indicating a high degree of similarity within the group. In contrast, the Karakatal population was positioned separately in the upper left quadrant, apart from the Kazakh Altai and Zailiysky Alatau. The Kaindysu population occupied an intermediate position between the central cluster and the northern populations (Akmola, Kostanay, and Karaganda). The Topkain population was the most distant population along the PCoA axis, highlighting its genetic distinctiveness. These findings were consistent with the AMOVA results and pairwise distance analysis, confirming the presence of a well-defined population structure within H. rhamnoides.

Figure 4 shows a phylogenetic tree based on the PBS profiling data of H. rhamnoides populations. Accordingly, the Shetlasty, Tersayryk, and Kendyrlyk populations formed distinct clusters. The Monyrak and Topkain populations demonstrated genetic isolation similar to that of the Kaindysu and Almaty populations. The central and northern Kazakhstan populations (Akmola, Karaganda, and Kostanay) were genetically close to each other but distinct from the other populations.

Bayesian clustering analysis was performed to assess the structure of Kazakhstani H. rhamnoides populations. The optimal number of genetic clusters was determined as described by Evanno et al. [80] The highest ΔK value was observed at K = 4 (Figure 5).

The Shelektsy, Karoiysu, Kendyrlyk, and Tersayryk populations were characterised by the dominance of specific clusters. In the populations from the central and southern regions of Manyrak, Karatal, and Almaty, a substantial cluster admixture was observed. The Akmola, Kostanay, Karaganda, and Topkain populations are predominantly represented by orange clusters, although traces of other genetic components were detected in certain cases.

4. Discussion

Sea buckthorn (H. rhamnoides L.) is a valuable perennial shrub of the family Elaeagnaceae with an extensive geographical distribution. Studying the genetic variability of sea buckthorn is important for breeding new cultivars with increased productivity, improved resistance to environmental stress factors, and high content of biologically active compounds [34,83].

Previous studies using various types of molecular markers (RAPD, ISSR, and AFLP) have revealed substantial within-population diversity, whereas interpopulation differentiation was relatively low and depended on the type of marker used.

The genetic diversity of a Kazakhstani sea buckthorn collection was studied for the first time using iPBS profiling. This marker system proved effective for detecting polymorphism without requiring prior genomic information, making it especially suitable for wild populations with limited background data [57].

Because iPBS markers are based on retrotransposons, which are activated under stress, they can reveal polymorphisms linked to adaptation [60]. This may explain why we were able to detect new loci and private amplicons in some populations, such as Topkain and Shetlasty.

Numerous studies examining sea buckthorn population diversity using various markers have revealed that diversity levels range from low to moderate [38,40,48,84,85]. Our results also showed that Kazakhstani sea buckthorn populations were characterised by a relatively low level of genetic diversity. This conclusion is supported by the values of the genetic diversity index I (0.169), He (0.113), and unbiased expected heterozygosity uHe (0.129), which may also be influenced by ecological factors. The iPBS profiling results indicated that the level of within-population variability (60%) exceeded that of among-population variability (40%), suggesting substantial differentiation of individuals within populations and relatively low genetic uniformity among different populations. A similar pattern of genetic diversity distribution, with a predominance of within-population variability, has also been revealed in geographically distant sea buckthorn populations using other marker systems (ISSR, SSR, and RAPD), which may be attributed to the species’ pollination and seed dispersal systems, as well as the influence of natural and anthropogenic factors [33,40,50,63,86].

Kazakhstani sea buckthorn populations growing in mountainous areas (Shetlasty, Kaindysu, Kendyrlyk, Tersayryk, Manyrak, Topkain, Karatal, and Almaty) and in forest-steppe and steppe zones (Kostanay, Akmola, and Karaganda) are influenced by diverse ecological factors and climatic conditions that are characteristic of Kazakhstan’s vast territory. Additionally, forest stands and mountain systems can considerably reduce the level of gene flow between populations, whereas the predominance of vegetative reproduction in wild sea buckthorn populations limits their genetic diversity.

The analysis of the Kazakhstani sea buckthorn populations indicated that Shetlasty and Tersayryk were genetically diverse and valuable from a breeding perspective. These populations were characterised by increased values of the effective number of alleles (Ne = 1.320 and 1.258), expected heterozygosity (He = 0.194 and 0.164), Shannon’s index (0.296 and 0.258), and proportion of polymorphic loci (PB = 60.0 and 59.1, respectively). The high levels of within-population diversity and balanced allele distribution in these populations may indicate a stable population structure, absence of pronounced isolation, and preservation of adaptive potential. This is further supported by the significant number of promising breeding forms and cultivars developed through adaptive selection from wild accessions of these populations [8].

In contrast, the Karatal and Topkain populations exhibited the lowest values of Na (0.617 and 0.548, respectively), Ne (1.116 and 1.061, respectively), and I (0.099 and 0.053, respectively), which may indicate a significant narrowing of the gene pool. This may be attributed to the predominance of vegetative reproduction (via root suckers), which limits recombination and formation of new genotypes, or to substantial population isolation. According to Vdovina, no single form of breeding interest has been identified in the Topkain population over more than 30 years of research. This population was characterised by the absence of large-fruited forms and the dominance of male individuals in the thickets. However, our study revealed two private amplicons in the Topkain population, indicating specific adaptations to local habitat conditions and genetic distinctiveness (geographical or associated with the pollination system) that enabled unique mutations to become fixed in the gene pool. This finding has important implications for biodiversity conservation and breeding programs.

Despite the low genetic diversity in the Karatal population, which is located in unique ecological conditions, interdune depressions of the Karatal sands and valuable dwarf forms with sweet fruits have been identified. The limited gene pool of sea buckthorn in the Karatal population reduces the potential for the selective breeding of new cultivars. However, the survival of sea buckthorn under extremely arid conditions promotes the formation of adaptive xerophytic ecotypes, which may serve as donors of valuable agronomic traits such as dwarfism and sweet fruit.

Among the other studied populations, the medium level of biodiversity characteristics of the Kaindysu and Kendyrlyk populations (Shannon index 0.202 and 0.189, respectively, compared to an average of 0.169) indicated their relative genetic stability. This is also supported by the Ne/Na ratio ≥ 1, which indicates a relatively balanced distribution of allele frequencies, while the average level of heterozygosity uHe (0.148 and 0.129, respectively, compared to the species average of 0.129) suggests weak genetic isolation. This may have been due to vegetative reproduction, habitat fragmentation, or limited gene flow. Kendyrlyk populations are characterised by vegetative propagation through root suckers, with few seed-origin plants, owing to dense turf, which impedes seed germination [8]. Our results are consistent with the findings from studies on wild sea buckthorn populations, where the most isolated populations were characterised by high levels of biodiversity [45,84].

Our study demonstrates the potential of the proposed multilocus analysis method for assessing the diversity of Kazakhstani populations of H. rhamnoides. We believe that this method has the right to exist on par with other approaches, both multilocus (RAPD, ISSR, iPBS, etc.) and single-locus (SSR). This type of marker, alongside SSR markers, is widely used for the analysis of population polymorphism in many plants and, in several studies, is not inferior in resolution power to existing methods. iPBS markers are a robust and efficient option for detecting genetic polymorphism as they capture variation across multiple loci in different chromosomal regions using just one primer. By sampling numerous largely unlinked loci simultaneously, iPBS profiling delivers broader genome coverage and greater discriminatory power than typical single-locus PCR assays (e.g., SSRs). This enables faster, more comprehensive assessments of genetic diversity. While our study provides valuable initial data on sea buckthorn biodiversity in Kazakhstan, a possible increase in the number of iPBS primers in future analyses will likely improve the reliability and comprehensiveness of the results.

Overall, studies on the genetic diversity in Kazakhstani populations have confirmed the high adaptive capacity of H. rhamnoides to survive under diverse ecological conditions. Second, iPBS profiling of Kazakhstani sea buckthorn populations may be useful for the formation of gene bank collections, breeding programs, and the conservation of natural populations. In particular, there is a need for the in situ conservation of populations with high diversity levels (Shetlasty and Tersayryk, need to be preserved as sources of valuable cultivars. The Altai Botanical Garden has created the largest number of valuable cultivars from promising lines originating from these populations (Asem, Shetlastinka, Jantarnaja, Solnyshko, Yozhik, Plakuchaja, Jubilejnaja Kotuhova). Whereas for populations with low diversity (Karatal and Topkain), the possibility of ex situ conservation should be considered, as they are the most vulnerable.

As demonstrated in studies of genetic variability, maintaining high levels of intraspecific diversity can facilitate adaptive responses to climate fluctuations and promote overall fitness within populations [85]. Consequently, integrating these high-diversity populations into breeding programs could yield varieties with improved ecological and medicinal traits, thereby enhancing both conservation efforts and economic viability for local communities. To support applied breeding, future work should combine iPBS screening with higher-resolution genomic approaches (e.g., SNP genotyping or genotyping-by-sequencing) to enable genetic mapping and the identification of quantitative trait loci (QTL) associated with key agronomic and adaptive traits [7].

Thus, our results underscore the importance of conserving and rationally utilising the genetic resources of sea buckthorn, including measures aimed at maintaining and enhancing within-population diversity.

5. Conclusions

To the best of our knowledge, this is the first study on H. rhamnoides L. populations from different regions of Kazakhstan using iPBS markers that provides a comprehensive characterisation of their genetic diversity and structure. The analysis revealed a substantial contribution of both within- and among-population variabilities, confirming the high informativeness and efficiency of this method for assessing plant gene pools. The identified features of the population genetic structure demonstrated that the adaptive potential of the species was maintained through genotype diversity within populations, whereas interpopulation differentiation indicated limited gene flow between different regions of distribution.

Thus, the application of iPBS markers is effective in revealing the level of genetic polymorphisms and analysing the population structure of H. rhamnoides. These results highlight the necessity of conserving the gene pool of wild populations and their utilisation in the development of potential cultivars with high adaptability and economically valuable traits.