Genetic diversity assessment of walnut (Juglans regia L.) genotypes from inner Anatolia region Türkiye using ISSR and RAPD markers

Abstract

Common walnut (Juglans regia L.) is a globally important nut crop valued for its nutritional and economic benefits. Despite Türkiye’s importance as a walnut producer, molecular characterization of local genotypes, particularly from historically cultivated regions such as inner Anatolia remains limited. This study assessed the genetic diversity of 18 walnut genotypes from the inner Anatolia region, Türkiye using two molecular marker systems, including Inter-Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD). The polymorphic bands were scored to calculate Jaccard similarity coefficients. Genetic relationships were analyzed using UPGMA dendrograms and Principal Component Analysis (PCA). A total of 72 bands (54 polymorphic; 75.0%) were generated by nine ISSR primers, and 53 bands (42 polymorphic; 79.2%) by five RAPD primers. Both marker systems indicated moderate genetic diversity within the genotypes. Genotypes ‘G3’, ‘G6’, and ‘G9’ had identical banding patterns (similarity = 1.00), likely attributable to their proximate geographic origin and same propagation history, whereas ‘G12’ and ‘G14’ were identified as genetically different. Population structure analysis demonstrated consistent clustering patterns, revealing both homogeneous and admixed genotypes. The Mantel test demonstrated a strong correlation (r = 0.81, p < 0.05) between genetic matrices derived from ISSR and RAPD, verifying marker congruence. The results highlight the effectiveness of using ISSR and RAPD markers as an economical method for evaluating genetic structure in under-characterized germplasm. The identification of genetically different genotypes is crucial for conservation and breeding techniques aimed at enhancing traits and genetic resilience in walnuts.

Introduction

Walnut (Juglans regia L.) is a commercially important deciduous tree with extensive global cultivation for its nutritional and commercial value^1,2. Walnut kernels are rich in beneficial elements, including proteins, vital fatty acids, antioxidants, vitamins, and minerals, which substantially enhance human health and nutrition^2,3. Walnuts are a significant source of polyunsaturated fatty acids, particularly omega-3 and omega-6, which are associated with decreased cardiovascular diseases, enhanced cognitive capabilities, and anti-inflammatory effects^4,5. Furthermore, walnuts are rich in vitamin E, folate, potassium, magnesium, and B vitamins^6,7. Vitamin E functions as an antioxidant, safeguarding cells from free radicals, whilst B vitamins promote the well-being of the neurological system^8,9. These substances collectively safeguard the body against oxidative stress and diminish the likelihood of disorders such as diabetes. Hence, walnuts play an important role in healthy and balanced nutrition globally.

Global walnut cultivation is dominated by China, the USA, Iran, and Türkiye¹⁰. Türkiye is projected to produce over 425,000 tons¹¹ and has a strategic position owing to its varied climatic zones and substantial germplasm repository¹². Walnut demonstrates remarkable genetic diversity due to its broad geographical distribution and long history of cultivation¹³. Türkiye serves as a primary source of origin and variability for walnuts, with genetically diverse populations of seedling-derived trees that exhibit significant heterogeneity in productivity, nut traits, phenology, and stress tolerance¹⁴. Türkiye is estimated to have approximately 5 million native walnut trees, representing a valuable genetic reservoir with substantial phenotypic diversity in traits such as late bud break, cold hardiness, and resistance to diseases¹⁵. Native cultivars, particularly in Central Anatolia and Eastern locations like Sivas, have unique agronomic characteristics influenced by both natural and human selection. These cultivars/genotypes are significant for both breeding purposes and local adaptability to abiotic stress conditions¹⁶.

Understanding the genetic diversity of walnuts is crucial for conservation and improvement initiatives¹⁷. Morphological markers have traditionally been used for phenotyping; however, their reliability is constrained by environmental factors¹⁸. Molecular markers provide a more reliable method for evaluating genetic diversity¹⁹. Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD) are prevalent methodologies owing to their simplicity, cost-effectiveness, and capacity to identify polymorphisms without previous sequence information^20,21.

The ISSR markers target interspersed areas between microsatellites, uncovering polymorphisms across many loci and providing both reproducibility and comprehensive genome coverage²². The RAPD markers, despite being more sensitive to experimental conditions, are efficient in differentiating closely related genotypes^23,24. Their combined use offers contrasting perspectives on genome-wide diversity and population structure^25,26. The ISSR markers have effectively differentiated walnut genotypes from various geographic origins and revealed population structures that do not correspond with mountain range types, indicating intricate patterns of walnut spread and past migration²⁷. Numerous studies have established the superior efficiency of ISSR markers for walnut diversity^26,28–33evidenced by their elevated polymorphism information content (PIC), marker index (MI), and resolving power (Rp) relative to other marker systems.

The RAPD markers are extensively used for assessing genetic variation in walnuts because of their simplicity, cost-effectiveness, and ability to examine numerous samples without requiring existing genomic data^26,34–36. A study of walnut genotypes from the Northwestern Himalayan area, using 13 SSR and 20 RAPD markers, demonstrated significant genetic variation, with allele counts per locus varying from two to six for RAPD primers³⁶. The similarity coefficients varied from 12 to 79%, with a mean of 49%, indicating considerable genetic diversity within the examined group³⁶. Nevertheless, RAPD markers exhibit lower polymorphism rates than ISSR markers; yet, they are still effective for assessing genetic diversity, particularly when used with other marker systems.

Molecular markers have revolutionized the evaluation of genetic diversity, facilitating accurate characterization of plant germplasm and supporting efficient breeding, conservation, and management techniques^37,38. Recent studies have effectively utilized these markers to assess genetic diversity among walnut populations in Türkiye and globally^32,39,40. This study aimed to assess the genetic diversity of common walnut genotypes using molecular markers. Although Türkiye is acknowledged as a hub of walnut diversity and a prominent worldwide producer, the genetic characterization of native genotypes, particularly from Inner Anatolia, is inadequate. Inner Anatolia is a historically important agricultural region characterized by varied agro-climatic conditions; nonetheless, its walnut germplasm has been somewhat overlooked in recent genetic studies^14,33,41. Considering the increasing challenges from climate change and agricultural intensification, it is essential to characterize the genetic structure of these genotypes for both in situ and ex situ conservation, as well as for prospective breeding initiatives^32,39,40. Therefore, this study evaluated the genetic diversity of 18 walnut genotypes collected from inner Anatolia region Türkiye using ISSR and RAPD markers. The major aims of the study were to assess marker efficacy, genetic linkages, and conservation and breeding implications by analyzing genetic similarity using UPGMA dendrograms and Principal Component Analysis (PCA).

Materials and methods

Plant materials

Eighteen walnut genotypes naturally acclimatized to the Divriği area of Sivas province situated in the inner Anatolia region of Turkey were selected for this study. The permission to collect leaf samples was taken from the Ministry of Agriculture. The collection and use of young leaf samples of walnut in this study complied with all relevant institutional, national, and international guidelines and legislation. Young leaf samples were obtained from individual trees of all genotypes for molecular analysis. Comprehensive sample information of genotypes is given in Table 1.

Table 1.

Background information on the walnut (Juglans regia L.) genotypes collected from inner Anatolia region (Sivas/Divriği) and characterized for genetic diversity in the current study.

Genotype Code	Longitude °N	Latitude °E	Altitude (m)
G1	39.37769	38.11475	1150
G2	39.37733	38.11397	1161
G3	39.37747	38.11514	1173
G4	39.37689	38.11467	1039
G5	39.37636	38.11394	1065
G6	39.37669	38.11461	1071
G7	39.48389	37.73653	1060
G8	39.48406	37.73639	1085
G9	39.48478	37.73611	1051
G10	39.46844	38.09533	1055
G11	39.46900	38.09392	1043
G12	39.46942	38.09564	1061
G13	39.44775	37.74658	1067
G14	39.44689	37.74303	1047
G15	39.44283	37.74328	1070
G16	39.34136	38.08900	1081
G17	39.34319	38.08800	1091
G18	39.34303	38.08061	1095

Genomic DNA extraction

Fully expanded young leaves were collected from the genotypes, immediately frozen in liquid nitrogen and brought to laboratory for DNA extraction. Total genomic DNA was isolated from leaf tissues using the CTAB method⁴². The purity and concentration of the DNA were verified using 1% (w/v) agarose gels and the NanoDrop ND-1000 Spectrophotometer, and working dilutions were standardized to 50 ng/µL.

ISSR and RAPD amplification

An initial screening of 18 ISSR and 11 RAPD primers⁴³ was performed on five samples to determine their genetic relationships. Nine ISSR and five RAPD primers exhibiting unique polymorphic patterns; hence, these were used for all samples. A total of 14 primers were used in the study (Table 2). The PCR reaction mixture was prepared by using 4 µL of DNA (20 ng), 1 µL of primer, 10 µL of PCR master mix (Eco Tech, Cat. No: ET5), and 10 µL of dH₂O. A total of 20 ng of DNA was used in each 25 µL PCR reaction.

Table 2.

ISSR and RAPD primers used for PCR amplification to evaluate genetic diversity in walnut genotypes distributed in inner Anatolia region Türkiye.

ISSR Primers Code	DNA Sequences (5’−3’)	Tm oC	Ta oC	PCR amplification
UBC-808	5′-AGAGAGAGAGAGAGAGC-3′	52	49	94 °C–3 min 94 °C–1 min 48–53 °C 1 min 72 °C–1 min 72 °C–10 min 35 cycles
UBC-810	5′-GAGAGAGAGAGAGAGAT-3′	50	47
UBC-811	5′-GAGAGAGAGAGAGAGAC-3′	53	50
UBC-826	5′-ACACACACACACACACC-3′	52	48
UBC-831	5′-CTCTCTCTCTCTCTCTT-3′	50	48
UBC-834	5′-AGAGAGAGAGAGAGAYT-3′	52	50
UBC-836	5′-AGAGAGAGAGAGAGAGYA-3′	52	50
UBC-873	5′-GACAGACAGACAGACA-3′	48	46
UBC-880	5′-GGAGAGGAGAGGAGA-3′	55	53
	RAPD Primers Code
OPA-2	5′-TGCCGAGCTG-3′	34	32	94 °C–3 min 94 °C–1 min 30–32 °C 1 min 72 °C–1 min 72 °C–10 min 35 cycles
OPA-5	5′-AGGGGTCTTG-3′	32	30
OPA-7	5′-GAAACGGGTG-3′	32	30
OPA-18	5′-AGGTGACCGT-3′	32	30
OPA-20	5′-GTTGCGATCC-3′	32	30

Tm = melting temperature, Ta = annealing temperature.

The PCR protocol included an initial denaturation at 94 °C for 3 min, followed by 35 cycles including denaturation at 94 °C for 1 min, primer-specific annealing temperatures (Table 2) for 1 min, and extension at 72 °C for 1 min. A final extension was conducted at 72 °C for 10 min.

A 2% agarose gel prepared in Tris-borate-EDTA (TBE) buffer was used for the electrophoresis of the PCR products. The gel was stained with ethidium bromide and visualized using a UV transilluminator (Bio-Rad Laboratories, Inc., Hercules, CA, USA). There were four replicates for each genotype and primer.

Statistical analysis

The scoring methodology used a binary system, with a value of 1 indicating the presence of a band and a value of 0 denoting its absence. The study concentrated only on bands that were luminous, distinct, and well-separated⁴⁴. Diversity metrics, including the effective number of alleles, gene diversity, Shannon diversity index, and Nei’s genetic distance, were computed using POPGENE software version 1.32 (https://sites.ualberta.ca/~fyeh/popgene.html)⁴⁵. The average polymorphism information content (PIC) for each primer was calculated using the Eq. 1⁴⁶:

1

Where PIC denotes the polymorphic information content, fi signifies the frequency of band existence, and 1 – fi indicates the lack of the band.

A dendrogram using Jaccard’s similarity coefficients was constructed with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) in MVSP 3.22 software⁴⁷. The MVSP 3.22 software facilitated the creation of a genetic similarity matrix. A pairwise genetic distance matrix was produced with MVSP 3.22 software, and a bar plot graph was built with STRUCTURE software (https://web.stanford.edu/group/pritchardlab/structure.html). Furthermore, Principal Component Analysis (PCA) was conducted using MVSP 3.22 to evaluate the genetic correlations among walnut genotypes⁴⁷. The correlation was also computed among genotypes.

Results

Fourteen distinct primers from the UBC and OPA series were used in the study. These primers had differing GC content and melting temperatures (Tm), and showed different efficacy in identifying genetic diversity (Table 3). The highest polymorphism rate (100%) was recorded with primers UBC-826, UBC-831, and UBC-834, indicating their efficacy in distinguishing genetic variation. The UBC-880 primer had the lowest polymorphism rate at 46%. The UBC-836 primer had the greatest genetic diversity, indicated by a h value of 0.42. The UBC-826 primer exhibited the lowest diversity, with a value of h = 0.15. Likewise, the highest value of Shannon’s information index (I = 0.61) was observed for UBC-836, indicating a substantial degree of genetic information and variation linked to this primer. The smallest I value (0.22) was determined for UBC-826. The UBC-836 primer had the greatest polymorphic information content (PIC) score of 0.84, confirming it as the most informative and discriminatory marker. Other primers exhibiting elevated PIC values were UBC-831 (0.60), OPA-2 (0.61), and OPA-20 (0.56). The smallest PIC value (0.30) was observed for UBC-826, indicating that it offered less information for identifying genetic variation compared to the other primers. Primers like UBC-836, UBC-873, and OPA-2 were notable for their elevated polymorphism rates and genetic diversity, indicating their significant discriminating capability. Consequently, it is advisable to emphasize these primers in breeding programs and genetic resource conservation research.

Table 3.

Genetic diversity parameters of ISSR and RAPD markers used for the molecular characterization of 18 walnut genotypes.

Markers		GC%	Number of Bands		Diversity parameter
			Polymorphic	Total	P%	ne	h	I	PIC
ISSR	UBC-808	53	6	8	75	1.38	0.23	0.35	0.45
	UBC-810	47	10	11	91	1.46	0.28	0.42	0.56
	UBC-811	53	6	9	67	1.37	0.22	0.33	0.40
	UBC-826	56	4	4	100	1.25	0.15	0.22	0.30
	UBC-831	47	4	4	100	1.44	0.30	0.47	0.60
	UBC-834	41	4	4	100	1.35	0.21	0.33	0.44
	UBC-836	44	8	9	89	1.73	0.42	0.61	0.84
	UBC-873	50	6	10	60	1.64	0.38	0.57	0.72
	UBC-880	60	6	13	46	1.40	0.24	0.37	0.48
RAPD	OPA-2	70	8	10	80	1.56	0.33	0.49	0.61
	OPA-5	60	10	11	91	1.33	0.21	0.32	0.42
	OPA-7	60	10	12	83	1.37	0.23	0.37	0.45
	OPA-18	60	8	12	67	1.40	0.27	0.44	0.54
	OPA-20	60	6	8	75	1.47	0.28	0.41	0.56

ISSR marker analysis

A total of 72 bands were generated from nine ISSR primers, of which 54 were polymorphic, resulting in a polymorphism rate of 75.0%. Each ISSR primer produced an average of 8.00 bands. The UPGMA dendrogram based on ISSR markers revealed the lowest genetic similarity (0.743) between genotypes ‘G2’, ‘G12’, and ‘G14’ (Fig. 1). Conversely, genotypes ‘G3’, ‘G6’, and ‘G9’ displayed high genetic similarity (1.00), indicating identical banding patterns (Fig. 2). The ISSR results reveal that genotypes ‘G3’, ‘G6’, and ‘G9’ had a similarity coefficient of 1.000; nonetheless, this result requires careful interpretation. ISSR markers are inherently dominant and fail to differentiate between homozygous and heterozygous loci, which may result in the omission of cryptic genetic variation. Consequently, high similarity ratings may not accurately represent true genetic identity, and the possibility for underlying genetic differences cannot be ignored.

Fig. 1.

UPGMA dendrogram for genetic similarity among 18 walnut genotypes evaluated by ISSR markers.

Fig. 2.

Pairwise genetic distance matrix obtained from nine ISSR primers for the walnut genotypes included in the study.

Principal Component Analysis (PCA) based on ISSR data confirmed these results (Table 4; Fig. 3). The first four principal components of the PCA executed on ISSR data explained 48.06% of the total variability. Genotypes were not distinctly grouped into separate clusters, indicating limited genetic divergence within the population and corroborating the dendrogram structure.

Table 4.

Principal component loadings of ISSR and RAPD primers across the first seven principal components (PC1–PC4), along with cumulative variance explained.

Primers	PC1	PC2	PC3	PC4
UBC-808	0.015	−0.088	−0.108	0.019
UBC-810	−0.048	0.136	−0.092	−0.009
UBC-811	−0.099	0.055	−0.107	−0.02
UBC-826	−0.061	0.114	0.044	−0.060
UBC-831	0.0125	0.023	0.145	−0.36
UBC-834	−0.611	0.0015	0.014	−0.034
UBC-836	−0.0015	−0.002	0.021	0.0002
UBC-873	−0.051	−0.063	−0.02	0.017
UBC-880	−0.019	−0.049	−0.013	−0.044
Cumulative Variance (%)	17.7	28.84	39.31	48.06
OPA-2	−0.112	0.012	0.066	0.048
OPA-18	−0.018	−0.015	0.001	0.008
0PA-20	−0.041	−0.110	−0.074	0.147
OPA-5	−0.157	−0.120	−0.074	−0.093
OPA-7	−0.096	0.050	−0.068	0.053
Cumulative Variance (%)	28.73	40.90	51.06	59.91

PC = principal component.

Fig. 3.

Principal component analysis revealing the relationship between 18 walnut genotypes included in the study based on ISSR markers.

RAPD marker analysis

A total 53 bands were amplified using five RAPD primers among which 42 were polymorphic, yielding a polymorphism rate of 79.2%. The UPGMA dendrogram constructed from RAPD data showed the lowest genetic similarity (0.412) between genotypes ‘G3’ and ‘G14’ (Fig. 4). In contrast, genotypes ‘G6’ and ‘G18’ showed the highest similarity (0.939), suggesting a close genetic relationship (Fig. 5).

Fig. 4.

UPGMA dendrogram for genetic similarity among 18 walnut genotypes evaluated by RAPD markers.

Fig. 5.

Pairwise genetic distance matrix obtained from five RAPD primers for the walnut genotypes included in the study.

The PCA analysis using RAPD data mirrored these clustering trends, showing relatively compact grouping among genotypes (Table 4; Fig. 6), which again suggested low overall genetic differentiation. The first four principal components of the PCA executed on RAPD data explained 59.91% of the total variability.

Fig. 6.

Principal component analysis revealing the relationship between 18 walnut genotypes included in the study based on RAPD markers.

Population structure

The Q-matrix results from the STRUCTURE analysis (K = 4) on the data obtained from ISSR markers categorized studied genotypes into four separate genetic groups (Fig. 7). Cluster 1 consisted of genotypes that mostly derived genetic contributions from the yellow component and had a very uniform structure. Members of Clusters 2 and 3 exhibited significant genetic mixing, including contributions from both the blue and green components. Conversely, genotypes ‘G14’, ‘G15’, and ‘G18’, categorized under Cluster 4, had a predominance of the red component, indicating a unique genetic lineage. The results indicate the existence of both pure and heterogeneous genetic structures within the sampled material, providing significant sources of diversity for future breeding activities.

Fig. 7.

Population structure of 18 walnut genotypes at K = 4, determined using ISSR markers, illustrating the composition of genetic clusters.

The Q-matrix results from the STRUCTURE analysis (K = 4) on the data obtained from RAPD markers grouped 18 investigated walnut genotypes into four distinct genetic groups (groups 1–4) (Fig. 8). Cluster 1 consisted of individuals (e.g., ‘G2’ and ‘G4’) mostly defined by the yellow genetic component and displaying a uniform genetic profile. These genotypes are mostly linked to a single genetic pool and are believed to represent purer and more isolated genetic compositions. Conversely, individuals categorized in Clusters 2 and 3 (‘G5’, ‘G6’, ‘G9’, ‘G10’, ‘G11’, and ‘G12’) had mixed genetic contributions from the green and blue components, indicating potential genetic mixing among these genotypes. A significant level of variability was seen among these individuals. Cluster 4, including genotypes ‘G1’, ‘G3’, and ‘G13’–‘G18’, was primarily characterized by the red component, indicating a unique genetic group. These individuals are likely to possess a shared genetic ancestry or may have seen little gene flow. The results reveal the existence of both genetically pure and admixed individuals, highlighting the extensive genetic diversity of the collection. This diversity is crucial for future breeding initiatives, especially in the establishment of core collections, use of heterosis, and sustainable management of genetic resources.

Fig. 8.

Population structure of 18 walnut genotypes at K = 4, determined using RAPD markers, illustrating the composition of genetic clusters.

Comparative analysis of marker systems

Both marker systems (ISSR and RAPD) produced comparable patterns in dendrogram and PCA analyses. Genotypes such as ‘G3’, ‘G6’, and ‘G9’ consistently clustered together, while ‘G14’ showed significant divergence in both datasets. The congruence between ISSR and RAPD results indicates that both marker types are suitable for detecting genetic relationships in Juglans regia germplasm, although RAPD provided slightly higher polymorphism in this study. A Mantel test was used to evaluate the association between the genetic similarity matrices obtained from ISSR and RAPD data. The study demonstrated a significant positive correlation (r = 0.81, p < 0.05), indicating a strong alignment between the two marker systems for genotype grouping. This result supports the consistency and reliability of ISSR and RAPD markers in reflecting the genetic links among the studied walnut genotypes.

Discussion

The study revealed moderate genetic diversity among 18 walnut genotypes distributed in inner Anatolia region of Türkiye, as shown by polymorphism patterns derived from ISSR and RAPD markers. The observed polymorphism rates (75.0% for ISSR and 79.2% for RAPD) align with the ranges reported in earlier studies. Başak et al.²⁵ documented 72.39% polymorphism across several marker systems, while Sevindik et al.³⁹ observed 70.57% (ISSR) and 74.54% (RAPD) in genotypes from Western Türkiye. These data illustrate the efficacy of both marker systems in detecting chromosomal variation. The moderate diversity observed in the current study contrasts with the significant variability documented in Iranian walnut populations (Nei’s gene diversity: 0.13–0.24; Shannon’s index: 0.23–0.44)³¹. The present study identified reduced polymorphism percentages compared to previous investigations on Turkish and Iranian walnut genotypes. The difference has several explanations beyond adaptive divergence. Sampling techniques such as genotype counting, geographic representation, and the inclusion of wild or cultivated populations may significantly influence genetic variability. The amount and selection of primers, particularly their polymorphic potential and genomic target regions, significantly impacted the resolution of the marker system. Climate stresses and historical land use patterns in the studied areas may have reduced genetic diversity.

The limited genetic diversity and close clustering of several genotypes (e.g., ‘G3’, ‘G6’, ‘G9’) indicate a limited genetic basis in the studied genotypes, possibly attributable to geographical proximity, agricultural selection methods, and potential clonal reproduction. The clustering can be attributed to their close geographic origin and similar ecological conditions, which may indeed contribute to their genetic similarity. Comparable results have been reported for Indian^26,36 and Chinese⁴⁰ germplasm collections, where genotypes were grouped regionally owing to common ancestry or trade activities. Nevertheless, distinct genotypes such as ‘G14’ and ‘G12’ showed considerable genetic divergence. These outlier genotypes are valuable from a conservation and breeding perspective¹³ as maintaining rare alleles is extremely important for the development of resilient cultivars and long-term genetic improvement. Genotypes ‘G12’ and ‘G14’ may possess unique alleles or rare genetic combinations. This distinctive feature highlights their potential significance in broadening the genetic basis of breeding activities. When linked to advantageous characteristics, such as drought resistance, delayed bud emergence, or pathogen resilience, these genotypes may function as significant parental lines for the development of improved cultivars. Furthermore, their incorporation into core collections will improve the representation of genetic diversity, so facilitating the long-term conservation and sustainable use of walnut genetic resources.

The similarity between ISSR and RAPD results in this study confirms the argument that integrating several molecular markers improves the reliability of diversification assessments. This aligns with previous studies^22,25 recommending integrated marker techniques for enhanced resolution in walnut and other woody perennial crops. Conservation programs must emphasize the preservation of widespread genotypes to ensure production stability, as well as genetically different individuals to retain adaptive capacity. This dual strategy is consistent with Adıgüzel and Solmaz¹⁶ for the management of Türkiye’s plant genetic resources.

In the future, using high-resolution co-dominant markers like SSRs³³ or next-generation sequencing technologies (e.g., DArTseq, GBS) will further increase the comprehension of fine-scale genetic structure. Integrating molecular data with phenotypic and agronomic characteristics may enhance genome-wide association studies (GWAS) for features such as nut production, disease resistance, and climate adaptation⁴⁸.

Conclusion

This study highlights the utility of ISSR and RAPD molecular markers in revealing the genetic diversity and relationships among walnut genotypes from the inner Anatolia region, Türkiye. Both marker systems showed moderate levels of polymorphism and consistent clustering patterns, reflecting limited genetic differentiation within the genotypes. However, several genotypes (particularly ‘G14’ and ‘G12’) were genetically distinct and may represent important reservoirs of novel alleles. The findings suggest that local walnut germplasm in the inner Anatolia region is genetically narrow, likely due to clonal propagation and limited gene flow. This poses potential risks in the face of environmental stress and disease outbreaks, underscoring the need for targeted genetic conservation. Distinct genotypes (e.g., ‘G14’, ‘G12’) should be prioritized for in situ and ex situ conservation to safeguard genetic variability. Future research should include broader geographic sampling across multiple regions to capture the full spectrum of walnut genetic diversity. Incorporating SSRs, SNP arrays, or next-generation sequencing (e.g., DArTseq, GBS) can improve resolution and enable genome-wide association studies.

Author contributions

Y.Ç. participated in conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation, writing—review and editing, and visualization.

Data availability

The data will be available from the corresponding author on request.

Declarations

Ethical approval

Not applicable as the study is not a clinical trial and did not include any patients.

Competing interests

The authors declare no competing interests.