Introduction
Exploring the diversity of plant genetic resources is of paramount importance because it provides valuable insights into the breadth and depth of genetic variations within plant species. This knowledge is crucial for sustainable agriculture, biodiversity conservation and crop development (for cultivating crops with enhanced traits) (Govindaraj et al., Reference Govindaraj, Vetriventhan and Srinivasan2015; Begna and Begna, Reference Begna and Begna2021). Understanding genetic diversity allows us to identify unique genotypes, unravel adaptive mechanisms and harness the vast potential of plant genetic resources to address global challenges, such as food security, climate change and emerging diseases (Govindaraj et al., Reference Govindaraj, Vetriventhan and Srinivasan2015).
Centella asiatica, commonly known as Gotu Kola or Indian Pennywort, is a valuable medicinal plant widely used for centuries because of its remarkable therapeutic properties (Padmalatha and Prasad, Reference Padmalatha and Prasad2008; Mathavaraj and Sabu, Reference Mathavaraj and Sabu2021). Its traditional uses range from healing wounds and inducing anti-inflammatory effects to promoting cognitive function and reducing anxiety (Delbo and Calapai, Reference Delbo and Calapai2010; Govindaraj et al., Reference Govindaraj, Vetriventhan and Srinivasan2015). Limited genetic studies of C. asiatica pose a significant challenge in comprehending its genetic diversity and population structure, which are essential for the conservation, cultivation and genetic improvement of the species; this is of concern because the demand for natural remedies and functional foods continues to increase (Mathavaraj and Sabu, Reference Mathavaraj and Sabu2021; Tripathy et al., Reference Tripathy, Verma, Thakur, Chakravorty, Singh and Srivastav2022). In this study, we analysed the genetic diversity of C. asiatica collected from six different island regions in Korea, using genotyping-by-sequencing (GBS) analysis to investigate the inter-regional variations in the species.
Experimental
Plant materials and DNA extraction
Ninety-six C. asiatica samples were collected from six different island regions of Korea from September to October 2021 (Fig. 1 and online Supplementary Table S1). The collected C. asiatica leaves were freeze-dried for DNA extraction using a freeze dryer (MCFD8518, ilShinBioBase, Korea). DNA was extracted using the DNeasy plant mini kit (Qiagen, Germany), following the manufacturer's instructions. Their voucher specimens are currently deposited at the Honam National Institute of Biological Resources.
Figure 1. Collection site of Centella asiatica in Korea six islands and geographic depiction of ADMIXTURE for two clusters. Optimal K for ADMIXTURE is two based on 10 replicates of K 1–20.
GBS analysis
The GBS analysis for this study was conducted by Seeders, Inc. (Daejeon, Republic of Korea). The GBS library was generated using a standard analysis method (Elshire et al., Reference Elshire, Glaubitz, Sun, Poland, Kawamoto, Buckler and Mitchell2011) and sequenced using the Illumina HiSeq X Ten platform (Illumina, San Diego, CA, USA) with 150-nt paired-end reads. Sequence preprocessing and single-nucleotide polymorphism (SNP) detection were performed according to the process described by Ma et al. (Reference Ma, Yang, Jo, Kang and Nam2021). A total of 9183 SNP genotyping data points were obtained, and several measures of genetic diversity were calculated.
Data analysis
Six population genetics' statistics describing the genetic variations in the species were estimated using the R packages adegenet and hierfstat (Goudet, Reference Goudet2005; Jombart, Reference Jombart2008; R Core Team, Reference R Core Team2023). Analysis of molecular variance was used to compare the six populations using poppr (Kamvar et al., Reference Kamvar, Tabima and Grünwald2014). ADMIXTURE was used to estimate the genetic structures of the populations (Alexander et al., Reference Alexander, Novembre and Lange2009). The predefined genetic clusters (K) were set to 1–20, and 10 different models were selected for analyses (which were repeated 10 times). The optimal K value was determined based on the cross-validation error in the ADMIXTURE analysis. The genetic and geographic distances among the six populations were compared using the hierfstat package, to calculate the pairwise Weir and Cockerham Fst, and the geodist (v0.0.7) package was used to measure the geographic distances (km), resulting in a plot of geographic distance against Fst/1 − Fst, which could support the subsequent investigations of outliers (Padgham and Sumner, Reference Padgham and Sumner2021).
Discussion
We assessed the genetic diversity of C. asiatica using next-generation sequencing, for the conservation of native C. asiatica in Korean island regions. With the increasing awareness of various benefits of traditional herbal medicines, the demand for diverse C. asiatica in Korea has increased; C. asiatica is used as an ingredient in various cuisines and a component in pharmaceuticals and functional cosmetics (Ha et al., Reference Ha, Kwon, Kim, Jeong, Hwang and Lee2010; Choi et al., Reference Choi, Oh, Lee, Lee, Jeong, Lee, Chang and Park2021; Swarup et al., Reference Swarup, Cargill, Crosby, Flagel, Kniskern and Glenn2021). Several efforts are underway to explore the potential applications of the species; however, most of these studies (especially those conducted in China and India) are limited to functional evaluations (Mathavaraj and Sabu, Reference Mathavaraj and Sabu2021). According to the Korean Citation Index, among the 118 papers on C. asiatica published in Korea, with the exception of two papers on plant distribution, all others focus on functional evaluation. Previous studies indicate that a genetic understanding of target plants is necessary not only for the selection of superior resources and development of new varieties but also for their efficient conservation, cultivation and genetic enhancement (Mathavaraj and Sabu, Reference Mathavaraj and Sabu2021; Swarup et al., Reference Swarup, Cargill, Crosby, Flagel, Kniskern and Glenn2021).
Although the C. asiatica samples collected from the six Korean island regions exhibited relatively high genetic diversity, very low genetic differentiation was observed among their populations (Table 1, online Supplementary Tables S2 and S3). Furthermore, no significant isolation-by-distance patterns were detected (online Supplementary Fig. S1). The ADMIXTURE analysis revealed two distinct clusters, but a very low genetic differentiation was observed within these clusters (inter-population variation of 0.5%) (online Supplementary Table S2 and Fig. S2).
Table 1. Summary of the genetic diversity and differentiation of Centella asiatica at the collection sites
*N, number of samples; Ho, observed heterozygosity; Hs, within population gene diversity; Fst, fixation index; Fis, inbreeding Coefficient; Ia, the index of association; rbarD, the standardized index of association.
†* and ***, P < 0.05 and P < 0.001, respectively; ns, not significant.
C. asiatica is primarily found in tropical and subtropical regions, such as India, South Africa and Madagascar; owing to its natural habitat in warmer climates, it is likely to be susceptible to low temperatures (Yousaf et al., Reference Yousaf, Hanif, Rehman, Azeem, Racoti, Hanif, Nawaz, Khan and Byrne2020). There is a lack of information regarding the introduction pathways and distribution of C. asiatica in Korea; however, it is possible that some individuals adapted to the colder conditions in Korea, becoming dominant and spreading clonally. Our results suggest that all the analysed C. asiatica samples were likely to be clones, as indicated by the rBarD values in Table 1. Similarly, with respect to the genetic resources of Korean Camellia sinensis, Lee et al. (Reference Lee, Lee, Sebastin, Shin, Kim, Cho and Hyun2019) reported that some individuals adapted to the environment after migrating from the Yunnan Province of China.
We confirmed little genetic differentiation among the C. asiatica samples collected from the six island regions. However, additional studies are required to assess the genetic diversity of native C. asiatica in Korea. Furthermore, follow-up studies on the variations in the phytochemical content and bioactivity among individuals would allow for the selection of superior individuals and utilization of these plant genetic resources for cultivar development and other applications.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262123001090.
Acknowledgements
This work was supported by a grant from the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (HNIBR202101115).
