The genus Allium, comprising approximately 750 underutilized species (Fritsch and Friesen, 2002), is the largest within the monocot group and includes numerous economically important plants. The key species include onion (Allium cepa L.), garlic (Allium sativum L.), chives (Allium schoenoprasum L.), leek (Allium porrum L.), and bunching onions (Allium fistulosum L.) (Khade et al., 2022). In addition to these well-known crops, the genus also encompasses lesser-known species, such as A. altaicum, A. ramosum, A. chinense, and A. tuberosum etc. which are of interest for their potential contributions to biodiversity research. In addition to their agricultural value, Allium species hold significant ecological importance, with some listed in the Red Book of countries such as Mongolia, Russia, and China due to concerns over their conservation status (Mandakh et al., 2020).

Underutilized Allium species exhibit significant chromosomal diversity, ranging from diploid to highly polyploid forms. This remarkable cytogenetic variability reflects the genus’ complex evolutionary history and dynamic genomic architecture, with origins tracing back to regions of Asia and Europe. Over time, these lesser-studied species have adapted to a wide array of ecological niches, resulting in a rich spectrum of phenotypic traits and specialized adaptations. Investigating their chromosomal profiles and evolutionary trajectories not only enhances our understanding of Allium genomics but also supports crop improvement efforts and biodiversity conservation. Notably, underexploited Allium populations represent untapped reservoirs of genetic diversity, offering valuable traits for sustainable breeding and long-term genetic resource management.

To date, the genetic diversity of onions and related Allium species has been examined using various molecular markers, including RAPDs (Kutty et al., 2006; dos Santos et al., 2012), ISSRs (Monteverde et al., 2015; Sudha et al., 2019; Brahimi et al., 2022, 2024), and combinations of RAPD and ISSR (Sudha et al., 2019), RAPD and PCR-RFLP (Arifin et al., 2000), and RAPD and SSR (Mohapatra et al., 2023). Additional marker systems such as RFLPs (McCallum et al., 2001), AFLPs (Van Heusden et al., 2000; Simó et al., 2014), TRAP (Kisha and Cramer, 2011; Anandhan et al., 2015), SRAP combined with ISSR (Hancı and Paşazade, 2025), ILP (Gowd et al., 2023; Jayaswall et al., 2024), and SRAP (Khade et al., 2024; Mansour et al., 2020) have also been widely applied.

More recently, SSR and SNP markers have been extensively employed to assess genetic variation in Allium fistulosum (Yamashita et al., 2010), Allium mongolicum (Hu et al., 2022), and Allium cepa (Mahajan et al., 2009; Khar et al., 2011; Khosa et al., 2014; Jayaswall et al., 2019; Gupta et al., 2020; Lyngkhoi et al., 2021; Chalbi et al., 2023; Habsatou et al., 2024), among others. Studies incorporating STS and SNPs (McCallum et al., 2012; Scholten et al., 2016; Duangjit et al., 2013; Villano et al., 2019; Mallor et al., 2014; Hanci and Gökçe, 2016; Rivera et al., 2016; Damon and Havey, 2014; Chand et al., 2018; Labate et al., 2020; Sahoo et al., 2023) further highlight the growing utility of these high-resolution markers in understanding genetic variation. Among these, SSR markers have emerged as a preferred tool due to their high polymorphism, co-dominant inheritance, reproducibility, and cross-species transferability (Son et al., 2012; Wang and Zhang, 2022). These characteristics make SSRs effective for evaluating genetic diversity and population structure in plant species.

In addition to nuclear markers, the chloroplast genome characterized by maternal inheritance offers a powerful system for elucidating evolutionary relationships, phylogeography, and population genetics within and across Allium species. As a type of SSR marker, chloroplast simple sequence repeats (cp-SSRs) are particularly advantageous due to their high mutability, conservation, variability, co-dominant inheritance, and organelle-specific transmission (Ebert and Peakall, 2009). In Allium, cp-SSR markers have successfully revealed genetic diversity, population differentiation, and evolutionary relationships among closely related taxa (Jayaswall et al., 2023). They have also been used to detect historical demographic events, such as bottlenecks and genetic drift, which are critical for understanding population dynamics (McCauley, 1995). Strikingly, cp-SSRs contributed to onion breeding programs by facilitating targeted genetic analyses and facilitating the development of conservation strategies (Sharma et al., 2020).

Cp-SSR markers offer a non-destructive and efficient means for detecting subtle genetic variations in the chloroplast genome (Powell et al., 1995). Only a few studies have reported the use of cp-SSR markers in Allium species, such as Allium cepa L., Allium sativum L., and Allium paradoxum (M. Bieb.) (Jayaswall et al., 2022). The present study aims to investigate chloroplast genetic divergence, heterozygosity, allelic diversity, population structure, and genetic relatedness across 96 underutilized Allium species using 22 cp-SSR markers. By doing so, this research seeks to provide a foundation for the strategic use of these genotypes in future breeding programs and to guide the conservation of these critical plant resources in the face of ongoing environmental challenges.

Chloroplast SSR markers have proven to be highly informative in studies of plant genetic diversity, phylogenetics, and population structure because of their uniparental inheritance, low recombination rates, and conserved genomic context. The central focus of this study was the development and characterization of chloroplast simple sequence repeat (cp-SSR) markers in Allium fistulosum, with subsequent amplification testing across 96 underutilized Allium species. A total of 22 novel cp-SSR loci were identified and validated, revealing distinctive patterns in repeat motif distribution that contribute to our understanding of cp-SSR evolution and utility within the Allium genus. Our findings revealed that tetranucleotide repeats were the most abundant (45.45%), followed by dinucleotide repeats (27.27%). Notably, mononucleotide repeats were deliberately excluded due to their lower informativeness and susceptibility to sequencing errors and polymerase slippage, a strategy aligned with best practices in cp-SSR marker development (Li et al., 2020). This exclusion also avoided overrepresentation of polyA/polyT stretches, which are highly abundant but offer limited polymorphic information. Furthermore, hexanucleotide motifs were absent, consistent with previous cp-SSR studies where longer motifs are generally rare in chloroplast genomes.

Our results partly align with prior studies in Allium species. For instance, Jayaswall et al. (2022) reported the development of 22 cp-SSRs in Allium cepa and Allium sativum, and 15 cp-SSRs in Allium paradoxum. In contrast to our current findings, their work found that tri-nucleotide repeats were the most frequent motif type (50%), suggesting potential interspecific variation in repeat motif composition within the genus Allium. This difference could be attributed to genomic structural variation or differing evolutionary pressures across species. When compared to broader angiosperm studies, our cp-SSR motif composition is consistent with patterns observed in other taxa. For example, Guo et al. (2022) identified 139 cp-SSR loci across 11 tree peony plastomes, while Shukla et al. (2018) reported 21–25 cp-SSRs in various Vigna species (V. angularis, V. radiata, and V. unguiculata), and Huang et al. (2018) found 92 SSR loci across six Cupressaceae plastomes. In these studies, di- and tetranucleotide repeats also predominated, underscoring a conserved pattern of SSR distribution in chloroplast genomes across plant lineages.

Interestingly, our findings contrast with those of Feng et al. (2023) in Physalis angulata, where mononucleotide repeats were the most abundant (68.24%), followed by tetranucleotides (12.28%). The abundance of mononucleotide motifs in that study likely reflects a different analytical approach that included these repeats, which, while common, are typically avoided in marker development due to their lower polymorphic potential. Overall, the distribution of cp-SSR motif types in Allium fistulosum reflects both conserved and species-specific patterns observed across plant taxa. Our deliberate methodological choices such as excluding mononucleotide repeats support the development of highly informative, polymorphic, and stable markers, which are essential for downstream applications such as population genetics, phylogenetic reconstruction, and genetic diversity studies in Allium and related genera. This work contributes to the growing genomic toolkit for Allium research and supports future efforts in conservation and breeding of underutilized species. The high success rate of amplification (90.91%) and clear electrophoretic profiles of these cp-SSR markers demonstrate their robustness and reliability for genetic analysis. Similar success has been reported in other species, such as tree peonies, where 19 out of 21 cp-SSR markers amplified strongly (Guo et al., 2022). The cp-SSR markers developed here complement existing nuclear SSRs by capturing maternal lineage information, thereby enhancing the resolution of genetic diversity studies in Allium species. Moreover, these markers are expected to support broader applications in phylogeography, conservation genetics, and breeding. As more chloroplast genome sequences become available, the cross-transferability and expansion of cp-SSR marker sets will continue to facilitate species-specific and cross-species analyses (Huo et al., 2019; Feng et al., 2023). This study lays the groundwork for future genomic research and supports the strategic use of cp-SSR markers in Allium crop improvement and biodiversity assessment programs.

The cp-SSR marker analysis confirmed the effectiveness of the developed markers in revealing polymorphisms and genetic variation within Allium germplasm. The high rate of polymorphism (89.2%) and broad allele size range reflect the utility of these markers for studying genetic structure and relationships, especially in underutilized populations. The average PIC value (0.20) aligns with previous findings by Jayaswall et al. (2023), who reported PIC values ranging from 0.007 to 0.427 in Allium germplasm via chloroplast-derived SSR markers. Although slightly lower than in studies by Gowd et al. (2024) (PIC: 0.24–0.98; avg. 0.608), Mallor et al. (2014) (avg. 0.64), and Hanci and Gökçe (2016) (up to 0.7), the moderate PIC values in this study may be attributed to differences in genome source (chloroplast vs. nuclear SSRs), marker selection criteria, and the genetic backgrounds of the tested accessions.

Similar studies have reported varying PIC values, with Baldwin et al. (2012) and Rivera et al. (2016) reporting averages of 0.45 and 0.51, respectively. Lyngkhoi et al. (2021) reported 53 alleles using 145 SSR markers, with PIC values ranging from 0.219 to 0.715 and an average of 3.54 alleles per locus. Khar et al. (2011) reported PIC values ranging from 0.00 to 0.89 with 60 primers, detecting 54 alleles across 19 primers, with an average of 2.84 alleles per locus. These comparisons highlight the impact of population structure, genomic origin, and SSR motif type on marker informativeness. Highly polymorphic markers such as cp-SSR3 and cp-SSR14, with more than 80% polymorphism, offer strong potential for use in genetic mapping and diversity studies. Similar findings by Gowd et al. (2024), where 92 polymorphic loci were identified using 19 SSR markers across 95 Allium accessions, underscore the importance of SSRs in understanding genetic variation.

Jayaswall et al. (2023) further demonstrated the utility of chloroplast-derived SSR markers in A. cepa and A. sativum, reporting heterozygosity values ranging from 0.009 to 0.540 and PIC values ranging from 0.007 to 0.427. These markers offer a reliable platform for evaluating genetic relationships between underutilized and cultivated Allium species. The observed genetic diversity in underutilized Allium accessions holds critical value for crop improvement. Traits such as disease resistance, yield enhancement, and abiotic stress tolerance can be introgressed from underutilized relatives into cultivated backgrounds. Therefore, the conservation and characterization of underutilized Allium germplasm remains essential for the resilience and sustainability of breeding programs. Recent advances underscore the complementary role of cp-SSRs markers alongside genomic tools in exploring Allium genetic diversity and evolutionary history (Khosa et al., 2015; Jayaswall et al., 2019; Gowd et al., 2024). The integration of cp-SSR data with nuclear SSR and genome-wide SNP dataset will further enrich our understanding of the genetic makeup of Allium species and support targeted breeding and conservation strategies. The observed number of alleles (Na) and effective number of alleles (Ne) support the existence of moderate polymorphism across the cp-SSR markers used. Markers such as cp-SSR09 and cp-SSR10, which exhibited relatively higher number of alleles and diversity indices, are particularly useful in revealing genetic differences among Allium accessions. In contrast, monomorphic markers such as cp-SSR02, cp-SSR03, and cp-SSR11 are likely associated with conserved regions of the chloroplast genome, offering limited intraspecies resolution but potential value for interspecific or phylogenetic studies (Gowd et al., 2024; Karic et al., 2018).

Generally, low Ho values align with the uniparental (mostly maternal) inheritance and haploid nature of the chloroplast genome, as well as the self-pollinating behaviour of many Allium species (Lyngkhoi et al., 2021). Nevertheless, the high He and I values of cp-SSR10 demonstrate its potential for distinguishing diverse genotypes and tracking lineage relationships. These findings are consistent with previous cp-SSR studies in Allium, where allele numbers typically ranged between 2 and 5 (Lyngkhoi et al., 2021), and in other genera, such as Ziziphus (Huang et al., 2015). Compared with nuclear SSRs and EST-SSRs, cp-SSRs tend to be less polymorphic, because they are located in more conserved regions of the genome (Ricciardi et al., 2020; Baldwin et al., 2012; Khar et al., 2011). However, their high cross-species transferability and evolutionary stability make them ideal for phylogenetic studies and for characterizing maternal lineages.

The cross-transferability results indicate a broad genetic base within the genus Allium. High transferability rates in species such as A. viviparum and A. negianum suggest their close genetic relationships with other members of the genus and their potential utility in breeding programs. In contrast, lower transferability in accessions such as A. altaicum indicates possible genomic divergence or evolutionary distance. These patterns of allele sharing and divergence can be exploited for both germplasm conservation and introgression breeding strategies aimed at enhancing stress tolerance or other desirable traits. Overall, the cp-SSR markers were effective in evaluating genetic diversity, understanding evolutionary relationships, and identifying candidate accessions for conservation and breeding. The combination of highly polymorphic and conserved markers allows a dual-purpose application: detailed intraspecies diversity analysis and broader phylogenetic studies across underutilized and cultivated Allium species.

Owing to their uniparental inheritance and lack of recombination, chloroplast SSR markers are well-suited for studying genetic relationships, evolutionary history, and domestication processes in plants (Sharma et al., 2020a). In the present study, these markers effectively distinguished underutilized and cultivated Allium species into six well-defined genetic clusters. The genetic homogeneity of Cluster I highlights the close relatedness among A. tuberosum and its allies, likely due to shared ancestry and cultivation across similar ecological regions, which aligns with the findings of Jayaswall et al. (2023), who reported similar clustering patterns. Cluster II, composed of various underutilized accessions, including A. chinense and A. fragrans, reflects cytoplasmic similarity and ecological coherence, corroborating earlier findings by Gowd et al. (2024). The presence of both cultivated and underutilized species in Cluster III emphasizes its potential as a genetic bridge, with A. fistulosum and related species offering valuable traits such as disease resistance and abiotic stress tolerance. Cluster IV, comprising cultivated A. cepa genotypes, exhibited reduced diversity, a consequence of domestication and selective breeding, which is consistent with domestication bottlenecks observed by Jayaswall et al. (2023). Cluster V presented the greatest genetic divergence, harboring underutilized species adapted to niche environments such as high altitudes, in agreement with findings from Nanda et al. (2016) and Gowd et al. (2024), highlighting their value as reservoirs of unique alleles. Cluster VI, comprising A. hookeri, presented species-specific genetic uniformity, likely due to restricted geographical distribution and domestication. Despite their narrow diversity, the members of this cluster possess unique traits that are valuable for region-specific applications. The high genetic variation observed in Clusters III and V underscores the evolutionary potential of underutilized species, reaffirming the importance of integrating underutilized relatives into breeding programs to increase stress tolerance, disease resistance, and adaptability in cultivated Allium species.

Understanding population structure is critical for effective germplasm conservation, trait mapping, and breeding applications. The identification of three distinct genetic clusters among the 96 underutilized Allium accessions aligns with earlier findings by Jayaswall et al. (2023), who also reported three subpopulations using cp-SSR markers in Allium accessions. The moderate allele–frequency divergence (0.11) and varying intracluster distances observed in this study suggest both shared ancestry and independent evolutionary trajectories among the populations. The relatively high proportion of membership in pop3 (50.2%) suggests a broad and genetically diverse group, potentially encompassing accessions with mixed ancestry. In contrast, pop2, with a lower proportion (18.5%), may represent a more genetically uniform or isolated subset. The findings also correlate with those of Lyngkhoi et al. (2021), who identified two groups in 96 underutilized Allium accessions through STRUCTURE analysis and five groups via discriminant analysis, illustrating how methodology and marker type influence the resolution of population structure. Gowd et al. (2024) reported four clusters using nuclear SSRs, highlighting differences attributable to marker origin (chloroplast vs. nuclear). Similarly, Chalbi et al. (2023) reported population differentiation in Allium landraces based on accession type rather than phenotypic traits. These collective observations underscore the utility of cp-SSR markers in deciphering maternal lineage and cytoplasmic diversity, which are particularly important for breeding strategies involving cytoplasmic male sterility or other organelle-linked traits. Thus, marker-based population structure analysis not only facilitates an understanding of genetic diversity but also provides a valuable framework for selecting parental lines and managing Allium germplasm effectively.

Together, the PCA and heatmap analyses provided a nuanced understanding of the genetic diversity and structure within the underutilized Allium accessions. The separation along PC1 likely reflects deep evolutionary divergence, whereas PC2 captures more recent or subtle genetic differentiation. The broader dispersion observed in one cluster indicates high intragroup variability, potentially representing genetically diverse underutilized relatives with adaptive significance. Moreover, the tighter grouping of the second cluster suggests a subset of accessions with conserved genomic features, possibly shaped by shared ancestry or ecological adaptation. These observations are consistent with common patterns in plant population genetics, where variable levels of diversity are often observed within and among clusters (Spanoghe et al., 2020). Notably, the PCA-based clustering results corresponded well with the three subpopulations identified through STRUCTURE analysis, supporting the robustness and complementary nature of both methods. The overlapping zones in the PCA further corroborate previous studies reporting gene flow and admixture among Allium species (Xiong et al., 2022). The heatmap visualization further reinforced the PCA outcomes by graphically representing the levels of genetic similarity and divergence, with the dendrogram effectively grouping genetically close accessions. Similar integrative approaches have proven valuable in deciphering population structure and evolutionary relationships in Allium and other crop species (Anwar et al., 2017; Saina et al., 2023). Overall, these findings not only validate the genetic groupings but also emphasize the utility of multivariate and hierarchical clustering tools in germplasm characterization, aiding in the selection of genetically diverse and elite accessions for breeding and conservation programs.

In conclusion, this study has significantly advanced our understanding of the genetic diversity of underutilized Allium species through the development and application of novel chloroplast SSR markers. The identification of 22 cp-SSR motifs from the A. fistulosum chloroplast genome, with 20 markers exhibiting high polymorphism and stability, provides a robust toolkit for genetic analysis in Allium. The high level of polymorphism (89.2%) observed across 96 underutilized Allium species underscores the effectiveness of these markers in capturing genetic variation. The population structure analysis revealed three distinct genetic clusters, complemented by phylogenetic grouping into six major clusters, which offers valuable insights into the evolutionary relationships and genetic differentiation within the genus. These findings have important implications for Allium conservation strategies and breeding programs, highlighting the potential of underutilized germplasm as a reservoir of genetic diversity for crop improvement. This study demonstrates the utility of cp-SSR markers in revealing the complex genetic tapestry of Allium species, paving the way for precision-guided conservation efforts and the development of improved cultivars with enhanced traits such as disease resistance and stress tolerance.