Rhynchostylis orchids, belonging to the Orchidaceae family, are tropical plants that produce stunning waxy flowers arranged in long and elegant inflorescences. These plants are native to Southeast Asia, Myanmar, and Thailand and grow on trees as epiphytes. Rhynchostylis orchids are characterized by their long, dark green leaves and a distinctive scent that is common across the genus, regardless of the blooming season of district species. There are about 1,300 species and 180 to 190 different genera of orchids in Thailand including major tropical orchids such as Vanda, Rhynchostylis, Ascocentrum, and Aerides. These orchids make significant contributions to the Thai orchid industry, particularly as cut flowers and potted plants (Thammasiri 2016). Currently, 54% of the orchids produced in Thailand are exported, while the remaining 46% are consumed in the domestic market (Thammasiri 2014).

Rhynchostylis, commonly referred to as the foxtail orchid (like Aerides), derives its generic name from the Greek rhyncho, beak and stylis, column describing the beaked column exhibited in this genus. The World Checklist of Vascular Plants (WCSP) currently recognizes four species as Rhynchostylis gigantea (Lindl.) Ridl., Rhynchostylis retusa (L.) Blume, Rhynchostylis rieferi (Higgins) and Rhynchostylis cymifera Yohannan, J. Mathew & Szlach. Two subspecies of Rhynchostylis gigantea; Rhynchostylis gigantea subsp. gigantea and Rhynchostylis gigantea subsp. violacea (Lindl.) Christenson are also recognized (Teoh 2022). Two species of this genus grow in Thailand, namely R. gigantea and R. retusa. The flower colors of R. gigantea range from white to white with red-purple spots, red-purple, and orange. This genus is popularly grown as a potted plant and used as a parent for producing interspecific hybrids (Thammasiri 2016).

In Thailand, Rhynchostylis orchids show large diversity. Diverse genetic characteristics are observed among closely related species, particularly in the distinctive floral forms of Rhynchostylis retusa. However, surveys on Rhynchostylis and closely related species encounter difficulty in distinguishing between specific species based on only the characteristics of their appearance (Sumaythachotphong et al. 2020).

Phenotyping, biochemistry, and DNA diversity are all utilized in assessing the diversity of genetic resources but methods for evaluating diversity using phenotype and biochemical characteristics are not always reliable due to environmental influences, labor intensity, and associated costs(McNally et al. 2009). Assessing genetic diversity through DNA analysis is the approach commonly utilized due to its repeatability, stability, and reliability. Several DNA-based molecular marker techniques have been developed to analyze genetic diversity including the analysis of restriction fragment length polymorphism (RFLP) using restriction enzymes, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and single nucleotide polymorphism (SNP) detection via polymerase chain reaction (PCR) (Jeung et al. 2005; Navinder Saini et al. 2004). The reproducibility of RAPD and AFLP techniques presents limitations, and the markers they generate are dominant, leading to constraints in assessing relationships among genetic resources (Amiteye 2021). A single 16 to 25 bp primer is used in ISSR, targeting identical regions among microsatellites. These primers include a nucleotide anchoring motif that targets microsatellite regions comprising eight repeating dinucleotide units, six repeating trinucleotide units, or multiple repeating tetra or penta-nucleotide units (Jing-Yuan et al. 2018). Recently, ISSR markers have emerged as an alternative system with the reliability and advantages of microsatellites (SSRs) due to their high polymorphism, rapidity, simplicity, reproducibility, and cost-effectiveness (Pradeep Reddy et al. 2002). This technique has been widely used in studies on orchids for genetic fingerprinting (Pongsrila et al. 2014), species or variety identification (Sharma et al. 2013), genetic mapping (Gholami et al. 2021), genetic diversity (Lal et al. 2023), evolution and molecular ecology (Suetsugu et al. 2022), and hybridization analysis (Fajardo et al. 2014).

Thai orchids are good examples, showing how an ornamental crop can develop with different varieties in its natural habitat to become a major crop. The development of the Thai orchid industry has taken a long time, but it is now a high-income business and an important part of the national economy (Sumaythachotphong et al. 2020). This study focused on R. gigantea, which has grown wild for over a century in Wat Pa Mancha Khiri, Mancha Khiri District, Khon Kaen Province, Thailand. Over 4,000 Chang Kra wild orchids bloom in full splendor on the branches of about 280 tree species every year during January and February (Thongtham 2012). Rhynchostylis has a short blooming a period which makes more difficult to distinguish variations among varieties based only on their appearance. Being considered endangered inhabitants, Rhynchostylis species such as R. gigantea and R. retusa are reported as endemic in Southeast Asian countries, including Thailand, Laos PDR, and Myanmar. Among these species, R. gigantea is the most well-known, with the highest demand compared to other native orchid species (Anuttato et al. 2017). As such, these orchids have been the subject of conventional breeding projects aiming to establish the genetics of their economically desirable traits (Sumaythachotphong et al. 2020).

Eight orchid plants were selected for study: Rhynchostylis gigantea (Lindl.) Ridl., R. gigantea var. harrisonianum Holtt., R. gigantea (white with red-blotches flower), R. gigantea var. rubrum Sagarik, R. gigantea var. vivaphandhul, R. retusa (L.) Blume., Vanda coelestis (Rchb.f.) Motes and Aerides houlettiana Rchb. f. These species were chosen because they are endemic to Thailand, easily cultivated, widely available, and popular(Julsrigival et al. 2013). Notably, Rhynchostylis subjected to continuously decreasing genetic variability and thus listed in the Appendix-II of CITES (Jariyajirawattana et al. 2020).

In this study, we have focused on investigating the genetic relationship within the species R. gigantea, including wild varieties and commercially favored hybrid cultivars, and comparing them with closely related species V. coelestis and A. houlettiana, utilizing ISSR molecular markers. The results can serve as a basis for implementing strategies in the future aimed at conserving, sustainably utilizing, and hybridizing these orchids.

Plant materials

This study was carried out in the Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Mahasarakham University, Thailand. Six Rhynchostylis accessions, one Vanda genotype and one Aerides accession were surveyed and collected from various locations in Northeast Thailand, including both wild and cultivated areas (Table 1). The species R. gigantea was studied at Wat Pa Mancha Khiri, Mancha Khiri District, Khon Kaen Province, Thailand in its natural habitat. The orchids grow naturally on tamarind trees and identified plants were collected. The collection locations of R. gigantea and the other seven orchid plants, along with GPS coordinates and location altitudes presented in Fig. 1. Eight orchid samples were collected and grown in the greenhouse of the Department of Biology, Faculty of Science, Mahasarakham University. The visual appearance of the specimens as well as their corresponding names in the collection, general geographical distribution, and specific origin are presented in Table 1, Figs. 1 and 2.

Table 1 . Locations of Rhynchostylis sp., V. coelestis and A. houlettiana accessions collected from Northeast Thailand.

S. No.	Location	GPS Coordinates	Altitude	Species collected
1	Wat Pa Mancha Khiri, Mancha Khiri District, Khon Kaen Province	16°07'07.2"N 102°32'00.8"E	163.16 m	R. gigantea (Lindl.) Ridl.
2	Agricultural Technology Park, Faculty of Agriculture, Khon Kaen University, Khon Kaen Province	16°27'52.9"N 102°48'47.0"E	176.11 m	R. gigantea var. harrisonianum Holtt.
3	Agricultural Technology Park, Faculty of Agriculture, Khon Kaen University, Khon Kaen Province	16°27'52.9"N 102°48'47.0"E	176.11 m	R. gigantea (white witd red-blotches flower)
4	Mancha Khiri District, Khon Kaen Province	16°07'28.6"N 102°32'20.9"E	163.16 m	R. gigantea var. rubrum Sagarik
5	Keung Subdistrict, Muang District, Maha Sarakham Province	16°12'28.3"N 103°15'55.2"E	143.98 m	R. gigantea var. vivaphandhul
6	Agricultural Technology Park, Faculty of Agriculture, Khon Kaen University, Khon Kaen Province	16°27'52.9"N 102°48'47.0"E	176.11 m	R. retusa (L.) Blume
7	Kut bak Subdistrict, Kut bak District, Sakon Nakhon Province	17°04'18.5"N 103°49'04.9"E	204.92 m	V. coelestis (Rchb.f.) Motes
8	Suranaree Subdistrict, Muang District, Nakhon Ratchasima Province	14°52'13"N 102°00'22"E	227.03 m	Aerides houlettiana Rchb. f.

*Species identification was based on appearance (Sumaythachotphong et al. 2020).

Figure 1. Sample collection sites. Here, 1: Wat Pa Mancha Khiri, Mancha Khiri, Khon Kaen Province; 2, 3, 6: Khon Kaen University, Khon Kaen Province; 4: Mancha Khiri, Khon Kaen Province; 5: Muang, Mahasarakham Province; 7: Kut bak, Sakon Nakhon Province; 8: Muang, Nakhon Ratchasima Province.

Figure 2. Flowers of R. gigantea (A), R. gigantea var. harrisonianum (B), R. gigantea (white with red-blotches flower) (C), R. gigantea var. rubrum (D), R. gigantea var. vivaphandhul (E), R. retusa (F), V. coelestis (G), and A. houlettiana (H).

DNA extraction

A small piece (2 cm×2 cm) of fresh leaf from each sample was used for DNA extraction and ground into a fine powder using liquid nitrogen. Genomic DNA was extracted from the homogenized mixture using a commercially available kit (PureDireX Genomic DNA Isolation Kit (Plants), Thermo Fisher Scientific Co., Ltd.), following the manufacturer's instructions meticulously.

Quantification of the extracted genomic DNA was performed by running the dissolved DNA in 1.0% agarose gel in 1X TBE (Tris-Borate-EDTA) buffer alongside uncut λ DNA of known concentration. To prepare for ISSR analysis, the genomic DNA was diluted to a concentration of 50 ng⋅μL^-1 using nuclease-free H₂O (Invitrogen™, Thermo Fisher Scientific Co., Ltd.), maintaining optimal conditions for the experiment.

ISSR-PCR fingerprinting

Sixteen ISSR primers (University of British Columbia (UBC), Biotechnology Laboratory, Vancouver, BC, Canada) were used for PCR amplification to study the polymorphism and banding patterns (Table 2). Polymerase chain reaction (PCR) was conducted in thermal cyclers (Biometra TAdvanced, Bio-Active Co., Ltd.). The ISSR-PCR reaction was prepared as a 20 µl reaction mixture containing 1 µl template DNA (50 ng⋅μL^-1), 16.55 µl nuclease-free H₂O, 0.8 µl MgCl₂ (50 mM), 1X PCR buffer, 0.4 µl dNTPs (10 mM), 0.05 µl ISSR primer (100 pmol⋅μL^-1) and 0.2 µl Taq DNA Polymerase (5 U⋅μL^-1). The amplification program was 4 minutes preheating and initial denaturation at 94°C, then 40 cycles of 1 minutes at 94°C denaturation step, 1 minutes primer annealing at 51°C/52°C/53°C/54°C/55°C depending on the primer used, and 2 minutes extension at 72°C, with a final extension of 10 minutes at 72°C. The PCR products were kept at 4°C until gel electrophoresis. The amplified DNA fragments were then fractionated by electrophoresis on 1.0% (w/v) agarose gel with 1X TBE buffer. Electrophoresis was performed under a constant voltage of 100 V for 60-90 minutes. The size of each DNA band produced was estimated using a 100 bp DNA ladder RTU (Bio-Helix Co, Ltd). The gel electrophoresis results were visualized using Visafe Green Gel Stain (Vivantis^®, Malaysia), and photographed under an ultraviolet transilluminator. Sequences and optimized annealing temperatures of the 16 ISSR primers used in this study are presented in Table 2.

Table 2 . List of ISSR (Inter-simple sequence repeat) primers used to assess genetic diversity in this study.

No.	Primer	Sequence (5’ - 3’)	Annealingtemperature (°C)	References
1	PCP-1	GAC GAC GAC GAC GAC	55	Pant et al. (2024)
2	PCP-2	AGG AGG AGG AGG AGG AGG	55	Pant et al. (2024)
3	PCP-3	GTG CGT GCG TGC GTG C	55	Pant et al. (2024)
4	UBC 810	GAG AGA GAG AGA GAG AT	53	Tran et al. (2022).
5	UBC 811	GAG AGA GAG AGA GAG AC	53	Pathak et al. (2022)
6	UBC 813	CTC TCT CTC TCT CTC TT	53	Fajardo et al. (2014)
7	UBC 815	CTC TCT CTC TCT CTC TG	53	Itsuji et al. (2015)
8	UBC 817	CAC ACA CAC ACA CAC AA	52	Itsuji et al. (2015)
9	UBC 819	GTG TGT GTG TGT GTG TA	52	Itsuji et al. (2015)
10	UBC 820	GTG TGT GTG TGT GTG TC	54	Tikendra et al. (2021)
11	UBC 827	ACA CAC ACA CAC ACA CG	54	Tikendra et al. (2021)
12	UBC 834	AGA GAG AGA GAG AGA GT	52	Shukla et al. (2017)
13	UBC 835	AGA GAG AGA GAG AGA GYC	52	Pant et al. (2024) Pant et al. (2024)
14	UBC 848	CAC ACA CAC ACA CAC ARG	52	Pant et al. (2024)
15	UBC 888	BDB CAC ACA CAC ACA CA	51	Pant et al. (2024)
16	UBC 890	CTC TCT CTC TCT CTV HV	51	Pant et al. (2024)

UBC = University of British Columbia primer

B, D, H, R, V, Y = IUB code

Data scoring and analysis

The DNA profiling data generated by 16 ISSR primers for the eight investigated genotypes, including six Rhynchostylis accessions, one Vanda genotype, and one Aerides accession, were used for statistical analysis. Distinctive, independent, and reproducible amplified bands/alleles were scored as present (1) or absent (0) across all orchid genotypes for each primer. The data were entered into a binary matrix and subsequently analyzed using the computer package NTSYS-pc (ver. 2.1) (Rohlf 1992).

The polymorphism information content (PIC) value was calculated for each ISSR locus using the formula developed by Roldan-Ruiz et al. (2000) PIC = 2fi(1- fi), where fi is the frequency of marker bands which were present and 1-fi is the frequency of marker bands which were absent. The dendrogram was created by the UPGMA (Unweighted Pairgroup Method with Arithmetical Averages) method in the SAHN (Sequential, Hierarchical, Agglomerative, and Nested Clustering) module of NTSYS-pc 2.1 software.

Banding patterns and ISSR analysis

All plant samples were collected from various locations in the northeast of Thailand (Table 1). In this study, all extracted genomic DNA showed sufficiently good quality as PCR templates with ISSR primers. Sixteen ISSR primers examined displayed the appropriate banding pattern with DNA samples from six Rhynchostylis specimens, one Vanda accession, and one Aerides specimen, and were subsequently used for further analysis (Table 2). The sequences of ISSR primers were longer and bound to the conserved regions between SSR regions, meaning that this method had superior characteristics such as simplicity and high reproducibility. The ISSR method is more effective for distinguishing genotypes with high genetic similarity due to the high mutation rate commonly seen in ISSR loci (Verma et al. 2017).

The PCR amplification with 16 ISSR primers yielded 83 bands ranging from 208 to 1,223 bp across six Rhynchostylis accessions, one Vanda accession, and one Aerides specimen. Representative images of band profiles generated with two ISSR primer pairs in all orchid accessions are shown in Figs. 3A and 3B and Figs. 4A and 4B. Among the 16 ISSR primers, band numbers varied from 1 to 13, averaging 5.19 bands per primer, with a standard deviation of 3.49. Primer PCP-3 displayed the maximum 13 bands, while primer UBC 848 displayed the minimum 1 band, and 80 DNA fragments (96.39%) were polymorphic. The average percentage of polymorphic bands was recorded as 95.21%. The PIC values of these 16 primers varied from 0.05 (UBC 848) to 0.44 (PCP-3) (Table 3), with a mean average of 0.21.

Table 3 . Band variation and PIC values of the sixteen ISSR markers used in this study.

S.No	Primer	Total number of bands	Number of polymorphic bands	Number of monomorphic bands	PIC value	Polymorphism percentage	Product size (bp)
1	PCP-1	11	11	0	0.40	100	404-1155
2	PCP-2	7	7	0	0.29	100	540-1213
3	PCP-3	13	13	0	0.44	100	279-927
4	UBC 810	4	4	0	0.18	100	550-1150
5	UBC 811	5	5	0	0.22	100	275-879
6	UBC 813	4	4	0	0.18	100	540-1180
7	UBC 815	5	5	0	0.22	100	220-1223
8	UBC 817	3	2	1	0.14	66.67	721-960
9	UBC 819	2	2	0	0.09	100	796-960
10	UBC 820	2	2	0	0.09	100	560-776
11	UBC 827	5	5	0	0.22	100	618-1091
12	UBC 834	2	2	0	0.09	100	355-678
13	UBC 835	6	6	0	0.25	100	425-1060
14	UBC 848	1	1	0	0.05	100	556-953
15	UBC 888	10	9	1	0.37	90	245-1126
16	UBC 890	3	2	1	0.14	66.67	208-822
Total		83	80	3
Mean		5.19±3.49	5.00	0.19	0.21	95.21

Figure 3. DNA amplification results with ISSR primers UBC835 (A) and PCP-3 (B). Lane M represents 100 bp DNA ladder; Lane 1, R. gigantea; 2, R. gigantea var. harrisonianum; 3, R. gigantea (white with red-blotches flower); 4, R. gigantea var. rubrum; 5, R. gigantea var. vivaphandhul; 6, V. coelestis; 7, R. retusa and 8, A. houlettiana.

Figure 4. Band profile generated by ISSR primers UBC815 (A) and PCP-1 (B). Lane M represents 100 bp DNA ladder; Lane 1, R. gigantea; 2, R. gigantea var. harrisonianum; 3, R. gigantea (white with red-blotches flower); 4, R. gigantea var. rubrum; 5, R. gigantea var. vivaphandhul; 6, V. coelestis; 7, R. retusa and 8, A. houlettiana.

Genetic diversity

The results showed that the 16 ISSR primers generated 83 bands ranging from 208 to 1,223 bp. In the DNA fragments, the average polymorphic DNA bands were up to 95.21%. This was important information for the molecular study and showed that the primers provided more detailed information about the genetic makeup of the individuals being analyzed. The PIC value is an important parameter showing the effectiveness of specific primers in genomic characterization. In this study, the PIC values of the 16 ISSR primers varied from 0.05 (UBC 848) to 0.44 (PCP-3) (Table 3), with a mean average of 0.21. Therefore, all these primers were deemed suitable for genetic diversity study according to the classification of Botstein et al. (1980).

Genetic similarity and clustering analysis

A UPGMA dendrogram derived from the 16 ISSR data was generated to explore the genetic relationships among the eight orchid accessions. The calculated genetic similarity coefficients showed a large variation between samples from 0.383 to 0.914 (full data is presented in Table 4). The largest genetic similarity coefficient was 0.914 between accessions R. gigantea var. harrisonianum (Khon Kaen University, Khon Kaen Province) and R. gigantea var. rubrum (Mancha Khiri, Khon Kaen Province), while the lowest was 0.383 between accessions R. gigantea var. harrisonianum (Khon Kaen University, Khon Kaen Province) and R. retusa (Khon Kaen University, Khon Kaen Province). These results suggested that a high level of genetic diversity was exhibited among all the samples.

Table 4 . Genetic identity matrix among the six Rhynchostylis specimens, one Vanda accession, and one Aerides specimen based on the analysis of sixteen ISSR markers.

	S1	S2	S3	S4	S5	S6	S7	S8
S1	1.000
S2	0.568	1.000
S3	0.790	0.679	1.000
S4	0.630	0.914	0.716	1.000
S5	0.580	0.543	0.617	0.580	1.000
S6	0.568	0.481	0.556	0.519	0.765	1.000
S7	0.568	0.383	0.531	0.420	0.568	0.605	1.000
S8	0.556	0.395	0.543	0.432	0.580	0.519	0.667	1.000

Note: S1, R. gigantea; S2, R. gigantea var. harrisonianum; S3, R. gigantea (white with red-blotches flower); S4, R. gigantea var. rubrum; S5, R. gigantea var. vivaphandhul; S6, V. coelestis; S7, R. retusa and S8, A. houlettiana, respectively.

The result from the ISSR phylogenetic analysis was correlated with the morphological characteristics. The accessions were divided into three main clusters (I, II, and III) (Fig. 5). Cluster I comprised of four samples from Khon Kaen Province; R. gigantea, R. gigantea (white with red-blotches flower), R. gigantea var. harrisonianum, and R. gigantea var. rubrum. Cluster II included two accessions of Rhynchostylis and Vanda from Mahasarakham and Sakon Nakhon Provinces as R. gigantea var. vivaphandhul and V. coelestis, respectively while Cluster III comprised two samples including R. retusa and A. houlettiana from Khon Kaen and Nakhon Ratchasima Provinces, respectively. Accessions from the same location or nearby were grouped within the same cluster.

Figure 5. Unweighted pair-group method with arithmetic average clustering for six Rhynchostylis accessions, one Vanda and one Aerides accession based on sixteen ISSR markers.

Sixteen ISSR markers were used to assess the genetic diversity of six Rhynchostylis accessions, one Vanda genotype and one Aerides specimen. Each marker distinguished only a few accessions but using a multiple marker set simultaneously allowed for the assessment of genetic diversity across various genetic resources. The ISSR primer sequences were longer and bound to conserved regions between the SSR regions, demonstrate superior characteristics in simplicity and high reproducibility. ISSR is more effective for distinguishing genotypes with high genetic similarity due to the high mutation rate commonly seen in ISSR loci (Verma et al. 2017).

In this study, ISSR primers generated 83 bands, with average polymorphic DNA bands up to 95.21%. Results showed that the primers provided more detailed information about the genetic makeup of the individuals being analyzed. The PIC value is an important parameter showing the effectiveness of specific primers in genomic characterization. In this study, the PIC values ranged from 0.05 to 0.44, with a mean average of 0.21. With the highest PIC value of 0.44, the primer PCP-3 was determined to be appropriate for genetic diversity research. According to the classification of Botstein et al. (1980), PIC values ≥ 0.5 are deemed to provide very high information, 0.5> PIC ≥ 0.25 indicate moderate information, and PIC <0.25 suggest little information. Therefore, the moderate PIC values observed in this study may be attributed to the requirement for additional marker types and/or a greater number of markers. Indeed, the PIC value of primer UBC 848 was considered to be extremely low, suggesting the need to increase the number of primers used or choose other DNA markers such as RAPD, SSR, or SNP to gain more genetic information.

ISSR data were used to calculate genetic similarity coefficients, with results indicating significant diversity among the samples, ranging from 0.383 to 0.914. The highest values were observed among the accessions of R. gigantea var. harrisonianum (pure-white flower) and R. gigantea var. rubrum (solid-red flower). The high genetic similarity between the distinct color variants of both R. gigantea suggests a close genetic relationship, likely due to recent divergence or hybridization (Bock et al. 2023). In contrast, the lowest value between R. gigantea var. harrisonianum (pure-white flower) and R. retusa (white with pink spots) indicates their significant genetic divergence. The results showed that genetic variations within R. gigantea do not correlate with the phenotypic flower color germplasm present.

UPGMA dendrograms of eight related orchids were created using genetic similarity coefficients and NTSYS-pc 2.1 software. The clustering pattern observed in the dendrogram suggests a close genetic relationship among the orchid specimens, with Cluster I, comprising only four accessions of R. gigantea, indicating a high genetic similarity among specimens, particularly those from Khon Kaen Province. The results support previous findings regarding the high genetic relationship within R. gigantea populations, likely due to their close geographic proximity and the potential for gene flow among individuals within the same region (Hamlin et al. 2020). The presence of both R. gigantea var. vivaphandhul and V. coelestis in Cluster II suggests a moderate level of genetic similarity (0.481) between these two accessions, despite them from different location. Similarly, Cluster III, comprised of R. retusa and A. houlettiana specimens from different provinces, suggests genetic divergence between these species, likely influenced by geographic isolation and genetic diversity (Andriamihaja et al. 2021).

An increasing number of studies have used DNA markers to investigate the genetic relationships between orchid species. Parab et al. (2008) investigated the level of genetic variation among populations of A. maculosum, an epiphytic orchid from Goa, India, using RAPD and ISSR markers. A report on Dendrobium orchids concluded that RAPD markers outperformed SSR markers in phylogenetic analysis, as they more accurately correlated with the descriptive morphological characteristics (Basavaraj et al. 2020). Tran et al. (2022) utilized a combination of 10 RAPD and 10 ISSR markers to evaluate the genetic diversity of 20 jewel orchids that did not correspond to the taxa previously studied. However, the RAPD marker has a significant drawback due to its lack of reproducibility and susceptibility to experimental conditions (Konzen et al. 2017).

ISSR markers have been utilized for assessing genetic diversity and other purposes. Pathak et al. (2022) studied the regeneration competence of an endangered orchid, Vanda cristata Wall. ex Lindl. using leaf explants with five ISSR primers. Results showed a high degree of genetic stability to obtain true-to-type plantlets. The protocol developed in this study may be useful for conserving V. cristata. Lal et al. (2023) analyzed the genetic variability among natural populations of two endemic orchids, A. multiflora and R. retusa in Uttarakhand, India using ISSR markers.

Sixteen ISSR markers were selected for this study based on the primers utilized in earlier studies. The PCR results showed high polymorphism and excellent repeatability of the allele pattern. The study of genetic relationships across eight orchid specimens categorized them based on their sampling locations and flower color phenotypes. Our study provided useful information of DNA profile to assess the genetic relationships among six accessions of Rhynchostylis, along with one Vanda and one Aerides accession. The DNA profiles were utilized to assess the genetic information of orchid germplasm and to preserve wild and cultivated resources. Based on this study, further analysis can lead to the development of novel markers specific to orchid varieties. The results can also be utilized to select parent plants and contribute to the success of orchid breeding programs.

This study was financially supported by Mahasarakham University, Thailand. The authors thank the Department of Biology, Faculty of Science, Mahasarakham University for the facilities provided.