In Thailand,
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
Eight orchid plants were selected for study:
In this study, we have focused on investigating the genetic relationship within the species
This study was carried out in the Laboratory of Molecular Genetics, Department of Biology, Faculty of Science, Mahasarakham University, Thailand. Six
Table 1 . Locations of
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 | |
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 | |
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 | |
4 | Mancha Khiri District, Khon Kaen Province | 16°07'28.6"N 102°32'20.9"E | 163.16 m | |
5 | Keung Subdistrict, Muang District, Maha Sarakham Province | 16°12'28.3"N 103°15'55.2"E | 143.98 m | |
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 | |
7 | Kut bak Subdistrict, Kut bak District, Sakon Nakhon Province | 17°04'18.5"N 103°49'04.9"E | 204.92 m | |
8 | Suranaree Subdistrict, Muang District, Nakhon Ratchasima Province | 14°52'13"N 102°00'22"E | 227.03 m |
*Species identification was based on appearance (Sumaythachotphong et al. 2020).
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.
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
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
The DNA profiling data generated by 16 ISSR primers for the eight investigated genotypes, including six
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.
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
The PCR amplification with 16 ISSR primers yielded 83 bands ranging from 208 to 1,223 bp across six
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 |
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).
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
Table 4 . Genetic identity matrix among the six
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,
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;
Sixteen ISSR markers were used to assess the genetic diversity of six
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
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
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
ISSR markers have been utilized for assessing genetic diversity and other purposes. Pathak et al. (2022) studied the regeneration competence of an endangered orchid,
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
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.
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