Philippine Journal of Crop Science (PJCS) April 2015, 40 (1): 82-88 Copyright 2015, Crop Science Society of the Philippines
Research Note
Genetic Diversity and Geographic Dispersion in Thymus spp. as Detected by RAPD Markers Valiollah Yousefi1,2 , Abdollah Najaphy1* , Alireza Zebarjadi1 and Hooshmand Safari3 1
Department of Agronomy and Plant Breeding, Campus of Agriculture and Natural Resources, Razi University, Kermanshah, Iran; 2 Present address: Department of Plant Breeding and Production, Faculty of Engineering and Technology, Imam Khomeini International University, Qazvin, Iran; Center of Agricultural and Natural Resources Research, Kermanshah, Iran. *Corresponding author,
[email protected],
[email protected] Thyme, as an aromatic medicinal plant and a perennial and woody herb from Lamiaceae has commercial, pharmaceutical and perfumery potentialities. Thymus is taxonomically a very complex genus with a high frequency of hybridization and introgression among sympatric species, and some species of this herb are endemic to Iran. From the chemical point of view, important biochemical components such as thymol and carvacrol are known in thyme. In the present study, 13 Thymus spp. accessions collected from different geographic areas of Iran along with one accession from England (Thymus vulgaris) were analyzed by Random Amplified Polymorphic DNA (RAPD) markers using 20 primers to discover genetic polymorphism. A total number of 510 bands were detected from 20 RAPD primers, of which 483 (94.31%) were polymorphic, with an average of 24.15 polymorphic bands per primer. The size range of the amplified products was 200-4000 bp. UPGMA cluster analysis was carried out using Jaccard similarity coefficients based on RAPDs. The dendrogram obtained from the method classified the 14 thyme accessions into four major groups. Scatter biplot based on principal coordinate analysis (PCoA) also revealed four groups and confirmed the results of clustering method with some minor disagreements. The accessions were relatively grouped according to the location where they had been collected. The molecular variation assessed in the study could elucidate largely geographic dispersion of the thyme accessions, and in combination with biochemical characteristics, can be useful to improve the efficiency of selection and breeding programs.
Keywords: genetic diversity, medicinal plant, molecular polymorphism, RAPD markers, Thymus spp.
INTRODUCTION In the past, people depended completely on herbal medications or traditional medicines and used some wild plants for cometics and perfumery purposes. Nevertheless, in the recent years, medicinal plants have represented a primary health care source for pharmaceutical and perfumery industries. Specifically, for wild and cultivated medicinal plants, there is a critical need for domestication, production, biotechnological studies and genetic improvement, instead of harvesting plants in the wild. The genus Thymus, a member of Lamiaceae/Labiateae family and an endemic species in Mediterranean, is comprised of numerous important aromatic species, which are commonly used as flavoring agents, herbal tea, and medicine. The aerial parts and volatile constituents of thyme, a perennial dwarf shrub, are used as a medicinal material (Stahl-Biskup & Saez 2002). In folk medicine, leaves and flowering parts of Thymus species are greatly used as tonic, antiseptic, antitussive and carminative as well as treating colds (Amin 2005; Ghasemi 2011). Among the Iranian species, T. Daenensis Celak and T. Kotschyanus Boiss and Hohen are more greatly used for these purposes (Zargari 1990; Nickavar et al. 2005). Recent researches have indicated that Thymus species have powerful antifungal, antibacterial, V Yousefi et al
antiparasitic, antiviral, spasmolytic, and antioxidant activities (Stahl-Biskup and Saez 2002; Nzeako and Al. Lawati 2008; Nabigol and Morshedi 2011). Forests and rangelands frequently experience fragmentation or isolation by human disturbance and thus suffer genetic bottlenecks. Severe bottlenecks, such as drastic reduction in population size, result in genetic erosion and loss of adaptation to environmental changes. In a small and isolated population, inbreeding depression as a result of mating between relatives diminishes the variability and viability of forests or rangelands. To keep relatively high genetic diversity within plant species, long-term genetic monitoring is needed (Nagata et al. 2005). Human interference in plant breeding is constantly directed to raise production, improve quality, and protect plants against pests. Some negative ecological effects of chemical usage and modern agricultural technologies restricted the number of genotypes. This latter view is especially critical because it causes genetic erosion, that is, a decrease in the species gene pool (Han et al. 2007). Also, Changes in agricultural systems and substitution of more profitable and exotic germplasm in place of traditional species were pointed out by Hammer et al. (1996) as the main causes of genetic erosion in landraces of several crop species in
Table 1. Accessions of Thymus spp. collected from different parts Accession code 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Gen bank code (RIFR) 18209 21118 27471 27800 13206 27814 15656 27221 25951 7507 14245 10126 – 14287
Scientific name
Country
Province
Place of seed collection
T. daenensis T. kotschyanus T. kotschyanus T. kotschyanus T. kotschyanus T. kotschyanus Thymus sp. T. daenensis T. kotschyanus Thymus sp. Thymus sp. T. daenensis Thymus sp. T. vulgaris
Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran Iran England
Isfahan Yazd West Azarbaijan Ardabil Gilan Ardabil Markazi Isfahan Kurdistan Lorestan Lorestan Isfahan Ardabil London
Buin-Daran Galuyak farm – Nedushan Sardasht Sarein Deilaman – Siahkal Pars Abad e Moghan Shahrak e Mohajeran Chadegan Ghorveh Khoram Abad – Zagheh Khoram Abad Fereidunshahr Parchin village –
Albania and Italy. Progress in plant breeding needs a wide genetic basis. Knowledge of genetic variation in crop species and their wild relatives has a critical importance for crop improvement (Saeidi et al. 2008). Hence, the threat to the genetic diversity existing in wild populations due to over-harvesting or habitat disturbance and the need for preservation of the genetic resources generates an encouragement to determine the genetic variability of Thymus species to guarantee its sustainable usage. A wide range of marker systems is now accessible for genotyping plant genomes. Markers are employed not only in plant breeding research, but also in applied plant breeding (Nagata et al. 2005). DNA fingerprinting techniques have been used widely for the analysis of the genetic diversity and to differentiate species or populations in plant conservation management systems (Morell et al. 1995; Rodriguez et al. 1999). The detection of population differentiation may assist in the definition of adequate units for conservation, thus providing an appropriate focus for conservation management or monitoring. RAPD has been broadly used for classification of accessions, identification of cultivars and genetic diversity evaluation (Beigmohamadi & Rahmani 2011). It is simple, less expensive, and rapid. It has the capability to detect extensive polymorphisms which require little amounts of genomic DNA even without prior knowledge of DNA sequences (Welsh and McClelland 1990; Williams et al. 1990; Clark et al. 1993). The present study was aimed to provide an overview of genetic variation and relationships among the Thymus accessions using RAPD markers.
MATERIALS AND METHODS Plant material A set of 14 Thymus spp. accessions was used for the analysis. A complete list of accessions and their origins is presented in Table 1. Ripe seeds of Thymus spp. was obtained from gene banking of RIFR (Research Institute of Forests and Rangelands), Iran in July 2010. Because of inability of thyme seeds to Genetic diversity of Thymus
germinate in soil, they were planted in glass vials under in vitro conditions, using the culture medium for seedling production (Murashige and Skoog 1962) medium. Aerial parts of thyme plantlets were collected and stored at -80°C. DNA extraction Genomic DNA from young leaves and stems was isolated using the CTAB (cetyltrimethyl ammonium bromide) procedure as described by Murray and Thompson (1980). Young plant tissues were crushed and powdered with liquid nitrogen in mortar. Ground tissues were immediately transferred to 1.5 mL Eppendorf tubes. DNA concentration was estimated spectrophotometrically, and the quality was checked by 0.8% agarose gel electrophoresis. The DNA samples were diluted to a working concentration of 5 ng µL-1. PCR amplification PCR amplifications were carried out in a CORBETT research thermal cycler, in a reaction volume of 25 µL containing 10X PCR buffer (2.5 µL), 2 mM MgCl 2 (1.4 µL), 0.2 µM primer (2.5 µL), 250 mMdNTP (0.4 µL), 1 U Taq DNA polymerase (0.2 µL) and 5 ng template DNA (1 µL). PCR cycling conditions for all accessions were 4 min initial denaturation (94°C); followed by 5 cycles of 30 s at 92°C, 60 s at 35°C, 90 s at 72°C, then 35 cycles of 5 s at 92°C, 40 s at 40°C, 90 s at 72°C, ending with a final extension step of 5 min at 72°C. All of the 20 RAPD primers (Table 2) produced a high number of bands and were used for the RAPD analysis of Thymus accessions. Amplified DNA fragments as PCR products were separated in a 1.2% agarose gel by electrophoresis and stained with ethidium bromide. The gel image was recorded using a Gel Documentation System (UVP, UK). Data analysis RAPD markers were scored for the presence (1) or absence (0) of amplified bands for each of 14 samples. The RAPD binary data matrix was used to calculate Jaccard similarity coefficient (Jaccard 1908). The binary matrix was used for statistical analysis using NTSYS-pc software version 2.02 (Rohlf 2000). 83
Table 2. Sequences of 20 RAPD primers used in this study Name of primer A7 AB1 C16 C9 E7 E10 E16 E17 E19 OPC07 OPC08 OPC10 OPC13 OPC15 OPC16 T9 T18 T19 U11 U17
Sequence of primer (5’-3’) GAAACGGGTG CCGTCGGTAG CACACTCCAG CTCACCGTCC AGATGCAGCC CACCAGGTGA GGTGACTGTG CTACTGCCGT ACGGCGTATG GTCCCGACGA TGGACCGGTG TGTCTGGGTG AAGCCTCGTC GACGGATCAG CACACTCCAG CACCCCTGAG GATGCCAGAC GTCCGTATGG AGACCCAGAG ACCTGGGGAG
For each RAPD marker, total amplified bands, number of polymorphic bands, percentage of polymorphic bands (PPB), polymorphism information content (PIC) and resolving power (RP) were recorded. Polymorphism information content (PIC) was calculated by applying the formula given by Powell et al. (1996) and Smith et al. (1997): where ƒi is the frequency of the ith alleles and the summation extends over n alleles. RP was calculated using the formula RP=∑Ib, where Ib is band informativeness and Ib= 1-[2×(0.5-p)], where p is the proportion of genotypes containing the band. Pair-wise genetic similarity between individuals for RAPDs was estimated using Jaccard similarity coefficient (Jaccard 1908). Cluster analysis using UPGMA (unweighted pair group method with arithmetic average) method (Sneath and Sokal 1973), and principal coordinate analysis (PCoA) were carried out by NTSYS-pc software version 2.02V (Rohlf 2000).
RESULTS AND DISCUSSIONS RAPD polymorphism The RAPD products ranged from 200–4000 bp (Figure 1). PCR amplification for the 14 accessions with the 20 primers generated a total of 510 clear and scorable bands, of which 483 were polymorphic (94.31%) and the rest were monomorphic (5.69%), which is comparable to some results in similar molecular studies of Thymus species and the other medicinal plants of the Lamiaceae family (Liu et al. 2006; Agostini et al. 2008; Rahimmalek et al. 2006). The polymorphism ranged between 83.33 and 100% (Table 3). The highest number of bands (37) was V Yousefi et al
obtained with primer U17 whereas E10 produced the lowest (11) (Table 3). An average of 25.5 amplified bands and 24.15 polymorphic bands was obtained per primer. Figure 1 shows the amplified bands using primer C16. The PIC values for the 20 primers varied from 0.28 to 0.48 with an average of 0.43. The lowest PIC indices were recorded for primers OPC15 and E17, and the highest PIC was obtained with U11 (Table 3). Sixty percent of the RAPD primers (12 primers) had PIC values more than the average showing highly informative markers used in this study. The estimates of RP (resolving power) varied from 5.14 (primer OPC15) to 24.14 (primer U17) with an average of 15.62 per primer (Table 3). RP was positively correlated with number of polymorphic bands (r= 0.85, P < 0.001) and PIC index (r=0.57, P < 0.01). Analysis of genetic relationships and variation Genetic relationships were pointed out by the range of Jaccard’s similarity coefficients that varied from 0.156 to 0.737 with the average of 0.280 indicating the high level of genetic variation that exists in the Thymus gene pool. The most genetically similar accessions were 4 and 6, with similarity coefficient of 0.737, and the least genetic similarity (0.156) was obtained between accessions 3 and 14. The cophenetic correlation coefficient between the original similarity matrix and the dendrogram was highly significant (r=0.884), showing the UPGMA clustering method was appropriate. Based on the cluster analysis (Figure 2), the thyme accessions were grouped into 4 clusters, whereby the genotypes in cluster 1 are more closely related than the individuals in different clusters. The first cluster was the largest, including 8 accessions, all three T. daenensis along with two T. kotschyanus and three Thymus sp. accessions. It was interesting to note that the first group included mostly the accessions collected from the center of Iran. Thymus vulgaris (London, England) was placed in the second cluster alone. Cluster three contained T. kotschyanus and Thymus sp. from Ardabil province. Two T. Kotschyanus accessions from north and northwest of Iran were placed in the fourth cluster. Scatter biplot based on principal coordinate analysis (PCoA) was used to illustrate the patterns of variation from the other point of view (Figure 3). The first two principle coordinates determined 25.78% of the total variation. PCoA also revealed four groups and confirmed the results of clustering method with some minor deviations. Group 1 contained all of the accessions classified in the first cluster except number 1 (T. Daenensis from Isfahan). Two accessions of T. kotschyanus from Ardabil province (4 and 6) were placed in a separate group. Accessions 1, 13 and 14 formed the third class. The fourth group (accessions 3 and 5 from north and northwest of Iran) in PCoA and clustering method was the same. Plant breeding is based on the identification and utilization of genetic variation. In recent years, a series 84
Figure 1. DNA fingerprinting of Thymus spp. using primer C16. M and C are size marker and control, respectively Table 3. Parameters of genetic variation generated by RAPD primers Primer A7 AB1 C16 C9 E7 E10 E16 E17 E19 OPC07 OPC08 OPC10 OPC13 OPC15 OPC16 T9 T18 T19 U11 U17 Total Maximum Minimum Mean
Total amplified bands
No. of polymorphic bands
PPBa
PICb
RPc
0.47 0.45 0.46 0.46 0.47 0.45 0.38 0.33 0.46 0.42 0.36 0.45 0.39 0.33 0.40 0.44 0.41 0.45 0.49 0.43 0.49
16.14 16.85 11 13.42 22.14 7.85 14.42 8.14 22.28 13.85 11.14 14 16.28 5.14 18.71 19.85 16.71 18.57 21.71 24.14 24.14
22 28 18 20 29 11 31 19 32 26 24 21 32 14 33 30 28 28 27 37 510 37
21 24 15 18 28 11 28 19 31 23 23 20 30 12 33 30 28 27 25 37 483 37
95.45 85.71 83.33 90 96.55 100 90.32 100 97 88.46 95.83 95.23 93.75 85.71 100 100 100 96.42 92.59 100 -
11
11
83.33
0.33
5.14
25.5
24.15
94.31
0.43
15.62
100
a
Percentage of polymorphic bands; b Polymorphism information content; c Resolving power.
of molecular markers, based on either proteins or DNA polymorphisms, have significantly facilitated research aimed at improving medicinal plant species. In the present study, RAPD markers created reproducible polymorphic bands in all of the 14 Thymus species, and provided a powerful and reliable molecular tool for detecting genetic diversity and relationships. The results of the analyses indicated that the accessions were relatively grouped according to location where they were collected. The relationship between genetic variation and geographic distribution has been reported in several species of aromatic Genetic diversity of Thymus
Figure 2. UPGMA dendrogram based on RAPD marker data
plants, for example, Tanacetum vulgare (Keskitalo et al. 2001), Artemisia annua (Sangwan et al. 1999), Thymus daenensis (Rahimmalek et al. 2009) and some other plants from Lamiaceae family (Fracaro & Echeverrigaray 2006; Liu et al. 2006; Agostini et al. 2008). As a result of existence of low similarity coefficients among most of the accessions, it can be concluded that there is a high genetic diversity among the accessions of Thymus. Accessions with the most different DNA profiles are likely to have the greatest number of novel alleles. Different geographical and ecological circumstances allow some possible genetic alterations or changes in DNA like translocation, deletion, point mutation and etc. The genetic diversity and relationships among Thymus species have been studied based on genotypic information. However, it is difficult to make comparisons between molecular studies, as different 85
Figure 3. Scatter biplot of 14 Thymus accessions using PCoA based on RAPD data genetic marker systems have been used on different populations. A number of studies using other marker systems suggest different evolutionary relationships. Dendrograms based on ISSR (Rahimmalek et al. 2009; Trindade et al. 2009; Smolik et al. 2009), RAPDs (Sunar et al. 2008; Alamdary et al. 2011; Trindade et al. 2009; Trindade et al. 2008), IGS-PCR (Smolik et al. 2009), and isozyme data (López-Pujol et al. 2004) indicated that all types of the markers were able to discriminate different accessions within Thymus spp. and generate polymorphisms.
CONCLUSION Our results have implications both for the conservation of natural genetic diversity and for the search toward novel sources of alleles to be used in thyme improvement programs. Since some indication of DNA polymorphism have been observed, a breeding program can be justified but the number of accessions should be expanded, to identify potential parents. A recurrent breeding and selection program could be mounted for specific biochemical components. The molecular diversity evaluated in the study in combination with morphologic and chemical characters can be useful to improve the efficiency of selection and breeding.
ACKNOWLEDGEMENT Financial support was granted by Razi University, Kermanshah, Iran.
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