Development and characterization of 15 polymorphic microsatellite markers for Hepatica insularis (Ranunculaceae) endemic to the Korean Peninsula

Article information

Korean J. Pl. Taxon. 2024;54(3):217-221
Publication date (electronic) : 2024 September 30
doi : https://doi.org/10.11110/kjpt.2024.54.3.217
Department of Biology Education, Chonnam National University, Gwangju 61186, Korea
1Department of Variety Examination, National Forest Seed Variety Center, Chungju 27495, Korea
2Department of Biological Sciences and Biotechnology, Chonnam National University, Gwangju 61186, Korea
3Division of Botany, Honam National Institute of Biological Resources, Mokpo 58762, Korea
Corresponding author: Jung-Hyun LEE, E-mail: quercus@jnu.ac.kr
Received 2024 August 12; Revised 2024 August 29; Accepted 2024 September 9.

Abstract

Hepatica insularis Nakai, a perennial herb endemic to warm-temperate regions on the Korean Peninsula, has been a subject of taxonomic debate due to its morphological similarities with Hepatica asiatica Nakai. To address the taxonomic ambiguity and provide evolutionary insights into H. insularis, we developed 15 polymorphic microsatellite markers. Of these, eight markers were successfully cross-amplified in H. asiatica, demonstrating their broader applicability. The markers exhibited high levels of polymorphism, with the number of alleles per locus ranging from two to eleven. The expected heterozygosity (HE) and observed heterozygosity (HO) values ranged from 0.180 to 0.802 and from 0.000 to 0.933, respectively. The developed markers will serve as a valuable tool for future phylogeographic studies aiming to understand the genetic structure and diversity of H. insularis and related taxa.

INTRODUCTION

A narrow zone extending from the western to the southern coast of the Korean Peninsula, encompassing approximately 3,000 islands, is characterized by the dominance of warm-temperate plant species (Lee and Yim, 2002; Jang et al., 2023). This distinct vegetation composition defines the area as the Southern Coast Floristic Province (Lee and Yim, 2002). Although the Jejudo Floristic Province, which has a similar climate, exhibits somewhat different flora, the majority of warm-temperate plant species on the Korean Peninsula are widely distributed in both regions. While both the Southern Coast and Jejudo Floristic Provinces are primarily warm-temperate, these regions display a subtle inclination towards subtropical conditions, as indicated by the presence of many plants typically associated with subtropical forests. For example, forests in these regions are predominantly composed of evergreen broad-leaved trees from the genera Machilus Nees, Neolitsea (Bentham & J. D. Hooker) Merrill, and Quercus L. [subgenus Cyclobalanopsis (Oersted) C.K. Schneid.] (Lee and Choi, 2010).

Within this context, Hepatica Mill., which comprises 11 taxa and is distributed across temperate regions of the Northern Hemisphere (Park and Park, 2021), includes H. insularis Nakai, an endemic species of the warm-temperate regions on the Korean Peninsula. This species, first described by Nakai in 1937, is particularly notable for its smaller size, which distinguishes it from H. asiatica Nakai, a species found in temperate regions on the Korean Peninsula and in cool-temperate regions of Manchuria (Park and Park, 2021). Aside from size, the two species exhibit few other distinct morphological differences, making their taxonomic separation challenging. Despite this, the status of H. insularis as a distinct species from H. asiatica has been the subject of ongoing debate among researchers. Kim and Lee (1994) and Woo et al. (2002) reported that, although the two taxa exhibit morphological similarities, genetic differentiation was observed through allozyme and isozyme analyses. However, Pfosser et al. (2011) found no clear genetic patterns that could distinguish the two taxa using nuclear AFLP and plastid marker analyses.

We speculate that the morphological and ecological differences between these two taxa may be influenced by various phylogeographic scenarios, as suggested by previous studies on plants in similar forest biomes (Lee et al., 2013, 2014; Han et al., 2020, 2023). To explore the evolutionary history of H. insularis, we developed 15 polymorphic microsatellite markers. We also tested the applicability of these markers to H. asiatica.

MATERIALS AND METHODS

To generate high-throughput sequencing data, we collected a fresh leaf sample of H. insularis from Jokeunnokkome Oreum, Jejudo Island, Korea, and extracted its genomic DNA using the DNeasy Plant Mini Kit (QIAGEN, Seoul, Korea) according to the manufacturer’s protocol. A voucher specimen (Voucher no. LeeHi190323V1) was deposited in the herbarium of Biological Education, Chonnam National University (BEC). The quality of the extracted DNA was evaluated using a Micro-Spectrophotometer Nano-300 (Allsjeng, Hangzhou, China), which showed a DNA concentration of 36.8 ng/μL, with A260/280 and A260/230 ratios of 1.94 and 2.22, respectively. A shotgun library was subsequently prepared using the Illumina MiSeq platform (LAS, Seoul, Korea), resulting in a total of 7,934,669 reads with paired-end sequencing (2 × 301 bp).

To identify microsatellites within these reads, we utilized the SSR pipeline v. 0.951 (Miller et al., 2013), configuring the settings to detect di-, tri-, and tetra-nucleotide motifs with flanking regions larger than 100 bp and containing at least 10, 6, or 4 repeats, respectively. Reads containing AT and TA repeats, those with GC content below 30% or above 80%, and reads with additional repeats were excluded. The sequences were categorized based on their repeat motifs and mapped to the reference genome using Geneious R 10.1.3 (Kearse et al., 2012). Contigs with sequence identities below 80% or quality scores under 50% were discarded. We selected sequences that exhibited unique patterns, had fewer than 20 reads, contained two distinct alleles, showed minimal variation at the primer attachment site, and had no additional single nucleotide polymorphisms in the flanking regions. These selected reads were deduplicated through de novo assembly and subsequently used for microsatellite marker development. We designed 90 primer pairs (18–22 bp in length) using the Primer 3 software within Geneious, targeting a melting temperature of 53–60°C and GC content of 35–65%. Forward primers were tagged with one of three M13 sequences (5′-CACGACGTTGTAAACGAC-3′, 5′-TGTGGAATTGTGAG CGG-3′, and 5′-CTATAGGGCACGCGTGGT-3′) and labeled with 6-FAM, VIC, or NED fluorescent dyes, respectively. For multiplex PCR, each primer set comprised nine primers ranging in length from 100 to 250 bp.

To assess the effectiveness of the newly developed microsatellite loci, we sampled 44 individuals of H. insularis from the Jjo and JS populations (Table 1). Cross-species amplification was also tested by sampling 28 individuals from the DG population of the related species H. asiatica (Table 1). PCR amplifications were performed in a final volume of 5 μL, containing 15–20 ng of DNA, 2.5 μL Multiplex PCR Master Mix (QIAGEN), 0.01 μM forward primer, 0.2 μM reverse primer, and 0.1 μM M13 primer (fluorescently labeled). The PCR protocol consisted of an initial denaturation at 95°C for 15 min, followed by 35 cycles of 95°C for 30 s, annealing at 56°C for 1.5 min, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. PCR products were analyzed using an ABI 3730XL sequencer with the GeneScan 500 LIZ Size Standard (Thermo Fisher Scientific, Waltham, MA, USA), and allele sizes and peaks for each sample were determined using Peak Scanner Software version 2.0 (Thermo Fisher Scientific). Genetic parameters, including the number of alleles, expected heterozygosity (HE), and observed heterozygosity (HO), were calculated with GenAlEx 6.5 (Peakall and Smouse, 2006). Deviations from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were evaluated using GENEPOP version 4.6.9 (Rousset, 2008).

Locality and voucher information for the Hepatica species used in this study.

RESULTS AND DISCUSSION

Using 90 newly designed primer pairs, we developed 15 polymorphic microsatellite loci, each exhibiting clear and strong peaks for every allele in 44 individuals of H. insularis from two populations (Table 2). The number of alleles per locus ranged from 2 to 11, with an average of 5.07. The 15 marker sequences developed for H. insularis were deposited in GenBank (MW092027-MW092045). The expected heterozygosity (HE) and observed heterozygosity (HO) values ranged from 0.180 to 0.802 and from 0.000 to 0.933, respectively (Table 3). Cross-amplification in the closely related species H. asiatica indicated that 8 loci were successfully amplified and were polymorphic, with 1 to 5 alleles per locus (Table 3). Although only one locus (HeI051) showed significant deviations from HWE after Bonferroni correction (p < 0.0033) across three populations, this deviation is likely due to population-specific factors rather than being marker-specific. Moreover, no significant LD was detected at any locus after Bonferroni correction, further supporting the reliability of the markers across the studied populations.

Characterization of 15 microsatellite loci for Hepatica insularis.

Genetic diversity parameters of 15 microsatellites developed for Hepatica insularis and cross-amplification in H. asiatica.

In conclusion, we developed a set of 15 polymorphic microsatellite markers from H. insularis and determined that 8 of these loci are applicable to the related species H. asiatica. The microsatellite markers described here will serve as a powerful genetic tool for elucidating the evolutionary patterns of H. insularis endemic to the Korean Peninsula. Furthermore, they will significantly contribute to advancing our understanding of both historical and contemporary gene flow between H. insularis and H. asiatica.

Acknowledgements

We sincerely appreciate the insightful feedback on Hepatica insularis from Dr. Jin-Kap Ahn (Jeonbuk National University). This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201905102).

Notes

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

References

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Article information Continued

Table 1.

Locality and voucher information for the Hepatica species used in this study.

Taxon Pop. ID Locality Geographic coordinates No. of individuals sampled Voucher No.
H. insularis Jjo Jokeunnokkome Oreum, Jejudo Island, Korea 33°23’46.7”N
126°24’53.2”E
29 LeeHi190323P1
H. insularis JS Mt. Naejangsan, Jangseong-gun, Jeollanam Province, Korea 35°27’41.0”N
126°50’33.8”E
15 LeeHi190525P1
H. asiatica DG Mt. Dodeoksan, Chilgok-gun, Gyeongsangbuk Province, Korea 35°59’26.6”N
128°36’17.0”E
28 LeeHi190511P1

One specimen per population was deposited in the herbarium of Biological Education, Chonnam National University (BEC).

Table 2.

Characterization of 15 microsatellite loci for Hepatica insularis.

Locus Primer sequence (5′–3′) Repeat motif No. of alleles Allele size range (bp) Fluorescent label GenBank accession No.
HeI002 F: TGGAATGTAAGAGTTGGAGT (TGT)6 4 150–165 6-FAM MW092027
R: GATCGGTCCGTTTACACTAA
HeI005 F: GCAGGATTTGTCAGTTTACC (AG)12 7 168–186 VIC MW092028
R: TTATTGCACTCATCATCGTG
HeI011 F: CAGCAATTAAAGGTATCTGATG (TG)10 6 190–200 6-FAM MW092031
R: TAATAGAAGGCTCCCATGTG
HeI016 F: CTTGATGTTGAACGTGGAAG (TTTA)5 3 152–160 NED MW092032
R: GCCCGTGGAATTAATTGATG
HeI025 F: TGATGTCAACACCAAAGTCT (ACA)6 5 153–174 NED MW092033
R: GTGATATACCGGATTGAGCC
HeI027 F: TGTTTCTTCAGTTGGTTCCT (GAA)6 3 260–266 NED MW092034
R: ATGATGGGGTTTGTTGGTAA
HeI044 F: CTCGATGAAGAACGCATAAA (TTG)9 5 205–217 NED MW092036
R: AGATCTGAGCTTTAAAACTAGG
HeI051 F: ACAGACTAGCCATGTTTTCA (TC)9 7 166–178 6-FAM MW092037
R: CAATCTCTTTATCGCCTCCA
HeI052 F: GGCTGTTGTGGCAATTTAAA (CCA)6 5 207–225 6-FAM MW092038
R: ATGTGATGAGGATGATGGTG
HeI055 F: GTCTTCTAAGCACGAGAGAG (AT)10 3 227–231 VIC MW092039
R: CTCTGTGTTGGCCTGAAATT
HeI104 F: CCTTTGTTTATTTCGGATTCCA (CTTT)3 3 116–124 VIC MW092040
R: CAGCTCTGCCATTCCCTC
HeI105 F: CGACCTGAATATTGATGCAA (GA)21 11 140–172 VIC MW092041
R: TGTGTCCAGAAAGTGTCAAA
HeI110 F: TCTTTAGTTGGAGATTTGGATC (CA)11 8 130–150 6-FAM MW092042
R: AGACATCTTCTTGAAAGTGGT
HeI114 F: CATGGTATGGTCTTTGGTCA (TGAT)4 2 203–207 VIC MW092044
R: GCATGCTCAAGTGATAAATCA
HeI115 F: TGAAGATTCGAAGGTGGTG (AAG)8 4 253–265 VIC MW092045
R: AATTTGTTTGGCGATAGGGA

Table 3.

Genetic diversity parameters of 15 microsatellites developed for Hepatica insularis and cross-amplification in H. asiatica.

Locus H. insularis H. asiatica


Jjo (N = 29) JS (N = 15) DG (N = 28)



A HE HO A HE HO A HE HO
HeI002 3 0.532 0.379 3 0.580 0.600 0 N/A N/A
HeI005 7 0.679 0.138* 2 0.278 0.067 0 N/A N/A
HeI011 5 0.738 0.241* 2 0.180 0.067 3 0.103 0.036
HeI016 2 0.366 0.483 2 0.498 0.933* 3 0.344 0.429
HeI025 2 0.452 0.345 5 0.720 0.000* 5 0.543 0.464
HeI027 2 0.498 0.310 3 0.551 0.133* 2 0.375 0.357
HeI044 5 0.612 0.310* 4 0.691 0.067* 0 N/A N/A
HeI051 6 0.729 0.103* 3 0.580 0.067* 3 0.534 0.964*
HeI052 4 0.400 0.276 3 0.287 0.067 0 N/A N/A
HeI055 3 0.380 0.207 3 0.518 0.267 5 0.658 0.571
HeI104 3 0.559 0.862* 2 0.420 0.600 3 0.513 0.929*
HeI105 10 0.769 0.241* 4 0.596 0.000* 0 N/A N/A
HeI110 5 0.532 0.310 7 0.802 0.267* 0 N/A N/A
HeI114 2 0.441 0.655 2 0.480 0.800 1 0.000 0.000
HeI115 4 0.537 0.897* 2 0.444 0.667 0 N/A N/A

N, number of individuals sampled; A, number of alleles; HE, expected heterozygosity; HO, observed heterozygosity; N/A, not applicable.

*

Significant deviation from the Hardy-Weinberg equilibrium after Bonferroni correction (p < 0.0033).