Method and primer for analyzing genetic diversity of rhizoma nardostachyos based on ISSR molecular marker

Abstract

The invention relates to a method and primers for analyzing genetic diversity of nardostachyos root and rhizome based on ISSR molecular markers, and belongs to the technical field of molecular biology. The first technical problem solved by the invention is to provide an ISSR molecular marker primer for genetic diversity analysis of rhizoma nardostachyos. The sequences of the primers comprise UBC809, UBC823, UBC840, UBC842, UBC847 and UBC850, and the UBC840, the UBC842, the UBC847 and the UBC850 are The six ISSR primer sequences provided by the invention are stable in PCR amplification reaction in rhizoma nardostachyos, clear in amplified fragment and relatively good in stability and repeatability. The method for analyzing the genetic diversity of the rhizoma nardostachyos based on the ISSR molecular marker provides technical support for analysis of genetic structures and genetic relationships of different geographical populations and different characters of the rhizoma nardostachyos, can complete identification of test materials in a low-cost, large-batch and short-time manner, and provides a basis for protection of rhizoma nardostachyos resources and breeding of excellent varieties.

Description

Technical Field

[0001] This invention relates to a method and primers for analyzing the genetic diversity of Nardostachys jatamansi based on ISSR molecular markers, and belongs to the field of molecular biology technology. Background Technology

[0002] Nardostachys jatamansi (D. Don) DC. is a perennial herb with a distinctive aroma, mainly distributed in high-altitude shrubs and grasslands in Tibet, Sichuan, Gansu, Qinghai, and Yunnan provinces of my country. Modern pharmacological studies have shown that it can be used to treat mental disorders, gastrointestinal discomfort, cardiovascular diseases, and skin problems. The combined use of Nardostachys jatamansi and levodopa is a promising treatment for Parkinson's disease, providing scientific evidence for subsequent clinical combination therapy with levodopa to enhance the anti-Parkinson's disease effect. Besides medicinal uses, it is also widely used in the fragrance, cosmetic, and food industries, and can be used to make incense, flavorings, and high-quality perfumes.

[0003] Due to long-term over-harvesting, habitat destruction, and low natural reproduction rates, the wild population of Nardostachys jatamansi has declined sharply and has been listed as a Class II protected plant in the "List of National Key Protected Wild Plants of China" (Second Batch). The depletion of wild resources not only threatens biodiversity but may also affect the sustainable development of related traditional Chinese medicine industries. Therefore, protecting Nardostachys jatamansi resources through multiple approaches, including rational resource development, domestication of wild plants, and breeding of superior varieties, has become a key research focus.

[0004] Molecular marker-assisted breeding technology, by directly analyzing the genetic information of the target species, can significantly improve breeding efficiency and is one of the core methods for improving the germplasm of Chinese medicinal herbs. Currently, the main DNA molecular markers used in molecular marker-assisted breeding research of Chinese medicinal herbs include SCoT, ISSR, SSR, and SNP. Among them, ISSR molecular markers have advantages such as low template DNA usage, simple operation, speed, reliability, and rich polymorphism, which help identify and determine the degree of genetic diversity of varieties, making it a very ideal genetic marker method.

[0005] The article "ISSR Analysis of Genetic Diversity of Forsythia Population" (Tang Zhenghui, Dai Ziwen. ISSR Analysis of Genetic Diversity of Forsythia Population [J]. Journal of Central South University of Forestry and Technology, 2025, 45(05): 132-140+153) discloses the use of ISSR technology to analyze the genetic diversity of Forsythia population. The article "Genetic Diversity Analysis of Tuberous Ardisia crenata Based on ISSR Molecular Markers" (Liang Hui, Deng Lili, Luo Baoli, et al. Genetic Diversity Analysis of Tuberous Ardisia crenata Based on ISSR Molecular Markers [J / OL]. Molecular Plant Breeding, 1-11) discloses the use of ISSR technology to analyze tuberous Ardisia crenata. The article "Evaluation of Genetic Diversity of Hainan Amomum villosum Germplasm Resources Based on ISSR and ITS Molecular Markers" (Luo Fan, Qu Yunping, Chen Zhenxia, ​​et al. Evaluation of Genetic Diversity of Hainan Amomum villosum Germplasm Resources Based on ISSR and ITS Molecular Markers [J]. Journal of Nuclear Agricultural Sciences, 2025, 39(05): 886-897) discloses the use of ISSR technology to analyze Hainan Amomum villosum. It is evident that ISSR molecular marker technology is currently used to analyze Chinese medicinal herbs such as Forsythia suspensa, Ardisia japonica, and Amomum villosum. However, despite the excellent performance of ISSR technology in other Chinese medicinal herbs, there is still a significant gap in research on Nardostachys japonica.

[0006] Lack of genetic diversity data: Existing studies have mostly focused on ecological adaptation or chemical composition, and the genetic background at the molecular level has not yet been systematically analyzed.

[0007] Shortage of specific primers: There is currently no publicly available ISSR primer library for Nardostachys jatamansi, which limits the direct application of this technology. Summary of the Invention

[0008] To address the above deficiencies, the first technical problem solved by this invention is to provide ISSR molecular marker primers for the analysis of genetic diversity in spikenard.

[0009] This invention provides ISSR molecular marker primers for analyzing the genetic diversity of *Nardostachys jatamansi*, the sequences of which include UBC809, UBC823, UBC840, UBC842, UBC847, and UBC850; wherein,

[0010] The sequence of UBC809 is: AGAGAGAGAGAGAGAGG;

[0011] The sequence of UBC823 is: TCTTCTCTCTCTCTCTCTC;

[0012] The sequence of UBC840 is: GAGAGAGAGAGAGAGAGAYT;

[0013] The sequence of UBC842 is: GAGAGAGAGAGAGAGAGAYG;

[0014] The sequence of UBC847 is: CACACACACACACACARC;

[0015] The sequence of UBC850 is: GTGTGTGTGTGTGTGTYC.

[0016] The second technical problem solved by this invention is to provide a method for analyzing the genetic diversity of Nardostachys jatamansi based on ISSR molecular markers.

[0017] This invention relates to a method for analyzing the genetic diversity of *Pharbitis nil* based on ISSR molecular markers, comprising the following steps:

[0018] 1) Extracting Nardostachys jatamansi DNA;

[0019] 2) The DNA extracted in step 1) was amplified using ISSR-PCR; the primers for the ISSR-PCR reaction system were the ISSR molecular marker primers for genetic diversity analysis of Nardostachys jatamansi as described in claim 1.

[0020] 3) Perform electrophoresis on the PCR amplification products after step 2), observe and photograph them using a gel imaging system;

[0021] 4) Analyze the electrophoretic bands obtained in step 3). Record the bands as 1 and the bands as 0. Create a 0-1 matrix and calculate the genetic distance or perform cluster analysis using software.

[0022] In one embodiment of the present invention, in step 1), DNA is extracted from dried Nardostachys jatamansi leaves using a plant genomic DNA extraction kit.

[0023] In one embodiment of the present invention, in step 2), the ISSR-PCR reaction system is as follows: each 20 μL reaction system contains 1 μL template DNA, 1 μL primer, 10 μL 2×Taq PCR Master Mix, and 8 μL ddH2O.

[0024] In a preferred embodiment, in step 2), the ISSR-PCR reaction program conditions are as follows: 94 °C pre-denaturation for 4 min; 94 °C denaturation for 30 s, 50 °C annealing for 30 s, 72 °C extension for 2 min, for a total of 34 cycles; 72 °C final extension for 5 min, and PCR amplification product stored at 12 °C.

[0025] In one embodiment of the present invention, in step 3), the electrophoresis is performed at 100V for 50 minutes in a 2% agarose gel.

[0026] In one embodiment of the present invention, in step 4), the electrophoresis bands are read and analyzed using Quantity One software, the genetic distance is calculated using POPGENE 32 software, and cluster analysis is performed using NTSYSpc 2.10e software.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention screened suitable primer sequences for Nardostachys jatamansi from 100 ISSR standard primer sequences published by the University of British Columbia (UBC), and optimized PCR conditions, ultimately obtaining 6 ISSR primer sequences. The PCR amplification reaction in Nardostachys jatamansi was stable, the amplified fragments were clear, and the stability and reproducibility were good.

[0029] This invention provides a method for analyzing the genetic diversity of *Nardostachys chinensis* based on ISSR molecular markers, offering technical support for analyzing the genetic structure and phylogenetic relationships of different geographical populations of *Nardostachys chinensis*. This method can identify experimental materials in a low-cost, large-scale, and short-term manner, providing a basis for the protection of *Nardostachys chinensis* resources and the breeding of superior varieties.

[0030] Genetic diversity analysis was conducted on 48 wild *Nardostachys chinensis* accessions using the method established in this invention. A total of 127 loci were amplified by ISSR-PCR, with 125 polymorphic loci, representing a polymorphism rate of 98.43%. The overall allele count (Na) of *Nardostachys chinensis* was 1.9843, the effective allele count (Ne) was 1.7113, Shannon's information index was 0.5784, and Nei's gene diversity (H) was 0.3977, indicating relatively rich genetic diversity. UPGMA cluster analysis was performed, and a dendrogram of genetic relationships among the samples was constructed. The results showed that the genetic similarity coefficient ranged from 0.51 to 0.75. When the similarity coefficient was below 0.53, the 48 samples could be divided into three categories, with the Tibetan samples forming a separate category. The results of this invention provide a scientific basis for the further development and utilization of *Nardostachys chinensis* germplasm resources and lay the foundation for understanding the genetic diversity of *Nardostachys chinensis* and its application in the breeding of high-quality varieties. Attached Figure Description

[0031] Figure 1 This is a DNA agarose gel electrophoresis detection image from Example 1.

[0032] Figure 2This is a PCR amplification diagram of 48 samples using primers UBC842 and UBC847 from Example 1. In the diagram, A: amplification diagram of primer UBC842; B: amplification diagram of primer UBC847; M is the DL 2000 DNA Marker; 1–12 are AB1–12; 13–24 are REG1–12; 25–35 are HY1–11; 36–39 are RT1–4; 40–43 are SD1–4; 44 is XZ; 45 is ML; and 46–48 are SP1–3.

[0033] Figure 3 This is a clustering result diagram of the ISSR molecular marker UPGMA of 48 Nardostachys jatamansi samples in Example 2 of the present invention. Detailed Implementation

[0034] This invention provides ISSR molecular marker primers for analyzing the genetic diversity of *Nardostachys jatamansi*, the sequences of which include UBC809, UBC823, UBC840, UBC842, UBC847, and UBC850; wherein,

[0035] The sequence of UBC809 is: AGAGAGAGAGAGAGAGG;

[0036] The sequence of UBC823 is: TCTTCTCTCTCTCTCTCTC;

[0037] The sequence of UBC840 is: GAGAGAGAGAGAGAGAGAYT;

[0038] The sequence of UBC842 is: GAGAGAGAGAGAGAGAGAYG;

[0039] The sequence of UBC847 is: CACACACACACACACARC;

[0040] The sequence of UBC850 is: GTGTGTGTGTGTGTGTYC.

[0041] Analyzing the genetic diversity of a species using only a single ISSR primer has limited accuracy. This invention discovers that using six specific ISSR primers to amplify Nardostachys jatamansi DNA and comprehensively analyze genetic diversity can improve the accuracy of the analysis results. The six ISSR primer sequences of this invention exhibit stable PCR amplification reactions in Nardostachys jatamansi, producing clear amplified fragments with good stability and reproducibility.

[0042] The second technical problem solved by this invention is to provide a method for analyzing the genetic diversity of Nardostachys jatamansi based on ISSR molecular markers.

[0043] This invention relates to a method for analyzing the genetic diversity of *Pharbitis nil* based on ISSR molecular markers, comprising the following steps:

[0044] 1) Extracting Nardostachys jatamansi DNA;

[0045] 2) The DNA extracted in step 1) was amplified using ISSR-PCR; the primers for the ISSR-PCR reaction system were the ISSR molecular marker primers for genetic diversity analysis of Nardostachys jatamansi as described in claim 1.

[0046] 3) Perform electrophoresis on the PCR amplification products after step 2), observe and photograph them using a gel imaging system;

[0047] 4) Analyze the electrophoretic bands obtained in step 3). Record the bands as 1 and the bands as 0. Create a 0-1 matrix and calculate the genetic distance or perform cluster analysis using software.

[0048] This invention provides a method for analyzing the genetic diversity of *Nardostachys chinensis* based on ISSR molecular markers, offering technical support for analyzing the genetic structure and phylogenetic relationships of different geographical populations of *Nardostachys chinensis*. This method can identify experimental materials in a low-cost, large-scale, and short-term manner, providing a basis for the protection of *Nardostachys chinensis* resources and the breeding of superior varieties.

[0049] Step 1) involves DNA extraction, which can be performed using conventional methods in the art. In one embodiment of the present invention, a plant genomic DNA extraction kit is used to extract DNA from dried Nardostachys jatamansi leaves.

[0050] Step 2) is the ISSR-PCR amplification reaction. The optimized ISSR-PCR reaction system of this invention is as follows: each 20 μL reaction system contains 1 μL template DNA, 1 μL primer, 10 μL 2×Taq PCR Master Mix, and 8 μL ddH2O.

[0051] The 2×Taq PCR Master Mix is ​​a premixed reagent designed to simplify polymerase chain reaction (PCR) operations. "2×" indicates a concentration twice the working concentration, and "Taq" refers to the thermostable DNA polymerase it contains. Its basic components include: Taq DNA polymerase, dNTPs (deoxynucleoside triphosphates), a buffer system, an enhancer / stabilizer, and a blue tracer dye. The 2×Taq PCR Master Mix can be commercially available. In one embodiment of the invention, the 2×Taq PCR Master Mix is ​​TIANGEN KT211-02 2×Taq PCR Premix Reagent II.

[0052] In a preferred embodiment, in step 2), the ISSR-PCR reaction program for primers UBC809, UBC823, UBC840, UBC847, and UBC850 is as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 50 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; final extension at 72 ℃ for 5 min; and storage of the PCR amplification product at 12 ℃. The ISSR-PCR reaction program for primer UBC842 is as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; final extension at 72 ℃ for 5 min; and storage of the PCR amplification product at 12 ℃.

[0053] Step 3) is electrophoresis. In one embodiment of the present invention, the electrophoresis is electrophoresis in a 2% agarose gel at 100V for 50 min.

[0054] In one embodiment of the present invention, in step 4), the electrophoresis bands are read and analyzed using Quantity One software, the genetic distance is calculated using POPGENE 32 software, and cluster analysis is performed using NTSYSpc 2.10e software.

[0055] The specific embodiments of the present invention will be further described below with reference to examples, but the invention is not limited to the scope of the embodiments described herein. The *Nardostachys jatamansi* samples used in the examples were wild *Nardostachys jatamansi* resources collected from the Sichuan-Western Plateau and Tibet, and identified as *Nardostachys jatamansi* (D. Don) DC., a plant of the Valerianaceae family. Fresh leaves of *Nardostachys jatamansi* were collected from the wild, dried in silica gel, and then used for later use. Detailed information is shown in Table 1.

[0056] Table 1 Sample Collection Information Table

[0057]

[0058] Example 1

[0059] 1. DNA extraction and detection

[0060] DNA was extracted from dried Nardostachys japonica leaves using a plant genomic DNA extraction kit (TIANGGEN DP305-02). Genome integrity was assessed by 1% agarose gel electrophoresis, and the concentration of the extracted DNA was determined using a micro-volume UV spectrophotometer (Rayli-Mettler-Toledo, UV5NANO, Switzerland).

[0061] The extracted DNA was analyzed and photographed using 1% agarose gel electrophoresis. The electrophoresis results showed that the DNA quality was good, and the electrophoretic bands were clear. Some results are shown below. Figure 1 As shown.

[0062] 2. Primer screening

[0063] The primer sequences for the ISSR reaction were synthesized by Beijing Qingke Biotechnology Co., Ltd. Preliminary screening of the primers was performed using mixed sample DNA. Primers that amplified multiple bands were further amplified against DNA from different origins to verify polymorphism. Finally, six primer pairs with clear bands and good polymorphism were selected. The sequences of the six selected primer pairs are shown in Table 2.

[0064] Table 2 Primer sequence listing

[0065]

[0066] Six selected primers were used to perform ISSR-PCR amplification on 48 samples of *Nardostachys japonica*. The ISSR amplification reaction system (20 μL) contained 1 μL template DNA (approximately 50 ng), 1 μL primers, 10 μL 2×Taq PCR Master Mix (TIANGENKT211-02 2×Taq PCR Premix II), and 8 μL ddH2O. The reaction program conditions for primers UBC809, UBC823, UBC840, UBC847, and UBC850 were as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 50 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; final extension at 72 ℃ for 5 min; and storage of PCR amplification products at 12 ℃. The ISSR-PCR reaction program for primer UBC842 was as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; final extension at 72 ℃ for 5 min; and storage of the PCR amplification product at 12 ℃. Detection: 5 μL of the PCR product was electrophoresed on a 2% agarose gel at 100 V for 50 min, and observed and photographed using a gel imaging system. The product bands were clear, and the stability and reproducibility were good. Some amplified bands are shown below. Figure 2 As shown. Figure 2 In the diagram, A represents the amplification pattern of UBC842 primers, and B represents the amplification pattern of UBC847 primers; M represents the DL 2000 DNA Marker, 1-12 are AB1-12, 13-24 are REG1-12, 25-35 are HY1-11, 36-39 are RT1-4, 40-43 are SD1-4, 44 is XZ, 45 is ML, and 46-48 are SP1-3.

[0067] The total number of statistical sites for the 6 primers was 127, with each primer generating 13 to 28 sites. Among them, 125 sites were polymorphic, accounting for 98.43% of the total sites. Except for primer UBC842, the percentage of polymorphic sites for the other primers was 100%. The primers used for ISSR molecular markers and the amplification results are shown in Table 3.

[0068] Table 3 ISSR Molecular Marker Primer Numbers and Amplification Results

[0069]

[0070] Example 2

[0071] DNA extraction was performed in the same manner as in Example 1.

[0072] Forty-eight samples of *Nardostachys japonica* were amplified by ISSR-PCR using the six primers listed in Table 1. The ISSR amplification reaction system (20 μL) contained: 1 μL template DNA (approximately 50 ng), 1 μL primers, 10 μL 2×Taq PCR Master Mix (TIANGEN KT211-022×Taq PCR Premix II), and 8 μL ddH2O. The reaction program for primers UBC809, UBC823, UBC840, UBC847, and UBC850 was as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 50 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; final extension at 72 ℃ for 5 min; and storage of the PCR amplification products at 12 ℃. The ISSR-PCR reaction program for primer UBC842 was as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; final extension at 72 ℃ for 5 min; and storage of the PCR amplification product at 12 ℃. Detection: 5 μL of the PCR product was electrophoresed on a 2% agarose gel at 100 V for 50 min, and observed and photographed using a gel imaging system.

[0073] The electrophoresis bands were read using Quantity One software, with bands marked as 1 and no bands marked as 0. A 0-1 matrix was constructed, and the genetic distance and phylogenetic tree were calculated using NTSYSpc 2.10e software, respectively.

[0074] Genetic distances of 48 samples were analyzed using POPGENE 32 software. The overall allele count (Na) of *Nardostachys chinensis* was 1.9843, the effective allele count (Ne) was 1.7113, Shannon's information index was 0.5784, and Nei's genetic diversity (H) was 0.3977, indicating relatively rich genetic diversity in *Nardostachys chinensis*. The ISSR primer combination screened using this invention can effectively distinguish differences in origin and traits of *Nardostachys chinensis*.

[0075] UPGMA clustering analysis was performed on Nardostachys jatamansi using NTsys 2.1e, and a phylogenetic tree was constructed. The results are shown in [link to results]. Figure 3 .

[0076] The phylogenetic tree results show that the genetic similarity coefficients range from 0.51 to 0.75, indicating high genetic diversity among *Nardostachys japonica* species. At a genetic similarity coefficient of 0.53, they can be divided into three groups: XZ forms a separate group; AB1-12, REG1-12, and HY1-4 cluster together; and HY5-11, RT1-4, SD1-4, SP1-3, and ML cluster together. This indicates that *Nardostachys japonica* DNA from Aba County, Ruoergai County, and Hongyuan County in Aba Prefecture has high genetic similarity, while Songpan County, also in Aba Prefecture, shows lower genetic similarity compared to other counties. Muli County in Liangshan Prefecture and Rangtang County in Aba Prefecture show high genetic similarity.

Claims

1. ISSR molecular marker primers for analyzing the genetic diversity of *Nardostachys jatamansi*, characterized in that: The primer sequences include UBC809, UBC823, UBC840, UBC842, UBC847, and UBC850; wherein, The sequence of UBC809 is: AGAGAGAGAGAGAGAGG; The sequence of UBC823 is: TCTTCTCTCTCTCTCTCTC; The sequence of UBC840 is: GAGAGAGAGAGAGAGAGAYT; The sequence of UBC842 is: GAGAGAGAGAGAGAGAGAYG; The sequence of UBC847 is: CACACACACACACACARC; The sequence of UBC850 is: GTGTGTGTGTGTGTGTYC.

2. A method for analyzing the genetic diversity of *Nardostachys jatamansi* based on ISSR molecular markers, characterized in that... Includes the following steps: 1) Extracting Nardostachys jatamansi DNA; 2) The DNA extracted in step 1) was amplified using ISSR-PCR; the primers for the ISSR-PCR reaction system were the ISSR molecular marker primers for genetic diversity analysis of Nardostachys jatamansi as described in claim 1. 3) Perform electrophoresis on the PCR amplification products after step 2), observe and photograph them using a gel imaging system; 4) Analyze the electrophoretic bands obtained in step 3). Record the bands as 1 and the bands as 0. Create a 0-1 matrix and calculate the genetic distance or perform cluster analysis using software.

3. The method for analyzing the genetic diversity of *Pharbitis nil* based on ISSR molecular markers according to claim 2, characterized in that: In step 1), DNA was extracted from dried Nardostachys jatamansi leaves using a plant genomic DNA extraction kit.

4. The method for analyzing the genetic diversity of *Pharbitis nil* based on ISSR molecular markers according to claim 2, characterized in that: In step 2), the ISSR-PCR reaction system is as follows: each 20 μL reaction system contains 1 μL template DNA, 1 μL primer, 10 μL 2×Taq PCR Master Mix, and 8 μL ddH2O.

5. The method for analyzing the genetic diversity of *Pharbitis nil* based on ISSR molecular markers according to claim 4, characterized in that: In step 2), the ISSR-PCR reaction program for primers UBC809, UBC823, UBC840, UBC847, and UBC850 was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 34 cycles; final extension at 72℃ for 5 min; and storage of the PCR amplification product at 12℃. The ISSR-PCR reaction program for primer UBC842 was as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 34 cycles; 72 ℃ final extension for 5 min, and storage of PCR amplification products at 12 ℃.

6. The method for analyzing the genetic diversity of *Pharbitis nil* based on ISSR molecular markers according to claim 2, characterized in that: In step 3), the electrophoresis is performed at 100V for 50 min in a 2% agarose gel.

7. The method for analyzing the genetic diversity of *Nardostachys jatamansi* based on ISSR molecular markers according to claim 2, characterized in that: In step 4), the electrophoresis bands were read and analyzed using Quantity One software, the genetic distance was calculated using POPGENE 32 software, and cluster analysis was performed using NTSYSpc 2.10e software.

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