• MATERIALS AND METHODS
• Plant materials and DNA isolation
• Primer screening
• PCR-RAPD
• Data analysis
• RESULTS
• Marker polymorphisms
• Genetic diversity
• DISCUSSION
• 적요
• ACKNOWLEDGEMENT

 Dill (Anethum graveolens L.) is a medicinal and aromatic plant belonging to the Apiaceae (Umbelliferae) family and is widely used as a spice and a medicine. The genus name Anethum  is derived from the Greek word aneeson or aneeton, which means “strong smelling”. Its common use in Ayurvedic medicine is for abdominal discomfort, colic, and promoting digestion. Dill is believed to be native to South-west Asia or Southeast Europe (Bailer  et al., 2001). It is indigenous to the Mediterranean, southern USSR, and Cen-tral Asia regions. Since Egyptian times, dill has been used as a condiment and also for medicinal purposes (Quer, 1981). It was used by Egyptian doctors 5,000 years ago and traces have been found in Roman ruins in Britain. In the Middle Ages, it was thought to protect against witchcraft. Greeks covered their heads with dill leaves to induce sleep. It grows up to 90 cm tall, with slender stems and alternate leaves, which are finally divided three or four times into pinnate sec-tions, slightly broader than the similar leaves of fennel. The yellow flower develops into umbels (Warrier et al., 1994). The seeds are not true seeds. The herb is a good companion when planted with corn, cabbage, lettuce, and onions, but inhibits the growth of carrots. Dill is used as an ingredient in gripe water, which is given to relieve colic pain in babies and flatulence in young children (Pulliah, 2002). The seed is aro-matic, carminative, and mildly diuretic, and used as a galact-agogue, stimulant, and stomachic (Hornok, 1992; Sharma, 2004). The essential oil in the seed relieves intestinal spasms and griping, helping to settle colic (Fleming, 2000; Duke, 2001). The carminative volatile oil improves appetite, relieves gas, and aids digestion. Chewing the seeds improves bad breath. Dill stimulates milk flow in lactating mothers, and is often given to cattle for this reason. It also cures urinary complaints, piles, and mental disorders (Nair & Chanda, 2007). Dill seeds are used as a spice and its fresh and dried leaves, called dill weed, are used as a condiment and in tea. The aromatic herb is commonly used for flavoring and sea-soning of various foods such as pickles, salads, sauces, and soups (Huopalathi & Linko, 1983; Blank & Grosch, 1991). Fresh or dried leaves are used to accompany boiled or fried meats and fish in sandwiches and in fish sauces. It is also an essential ingredient of sour vinegar. Dill oil is extracted from the seeds, leaves, and stems, which all contain the essential oil used as a flavoring in food industry. It is used in perfum-ery to aromatize detergents and soaps and as a substitute for caraway oil (Lawless, 1995). Dill is applied as a preserva-tive as it inhibits the growth of several bacteria, including Staphylococcus,  Streptococcus,  Escherichia coli, and Pseudomonas. Compounds of dill when added to insecti-cides have increased their effectiveness. Several experimen-tal investigations have been undertaken in diverse in vitro and in vivo models. Some pharmacological effects of  A. graveolens have been reported, such as antimicrobial (Chau-rasia & Jain, 1978; Delaquis et al., 2002; Nair & Chanda, 2007), antihyperlipidemic, and antihypercholesterolemic properties (Yazdanparast & Alavi, 2001).

 Recently, molecular methods have been used for the iden-tification and classification of different species of herbs and medicinal plants (Momeni et al., 2006; Solouki et al., 2008; Yu et al., 2011) and also for the evaluation of genetic diver-sity within and among species, as well as plant populations(Raghu  et al., 2007). These methods support the classic methods such as morphological and physiological traits(Dolezalova et al., 2003) and are suitable for genetic studies as environmental conditions do not affect them (Ali et al., 2007). DNA marker-based fingerprinting can distinguish species rapidly using small amounts of DNA and is there-fore able to deduce reliable information on their phyloge-netic relationships. DNA markers are not typically influenced by environmental conditions and therefore can be used to describe patterns of genetic variation among plant popula-tions and to identify duplicated accessions within germ-plasm collections. Various approaches are available for DNA fingerprinting such as amplified fragment length poly-morphism (AFLP) (Zabeau & Vos, 1993), restriction frag-ment length polymorphism (RFLP) (Botstein et al., 1980), simple sequence repeats (SSRs) (Tautz, 1989), and ran-domly amplified polymorphic DNA (RAPD) (Williams et al., 1990). Among these, RAPD is an inexpensive and rapid method that requires no information regarding the genome of the plant, and has been widely used to ascertain genetic diversity in several plants (Belaj et al., 2001; Deshwall et al., 2005). RAPD analysis requires only a small amount of genomic DNA; it can produce high levels of polymorphism and may facilitate more effective diversity analysis in plants(Williams  et al., 1990). RAPD analysis provides informa-tion that can help define the distinctiveness of species and phylogenetic relationships at the molecular level. Use of such techniques for germplasm characterization may facili-tate the conservation and utilization of plant genetic resources, permitting the identification of unique genotypes or sources of genetically diverse genotypes. RAPD analysis has been used for genetic diversity assessment and for iden-tifying the germplasm in many plant species (Welsh & McClelland, 1990; Kapteyn & Simon, 2002). The aim of this study was to select RAPD markers for investigating genetic diversity in dill species.

MATERIALS AND METHODS

Plant materials and DNA isolation

 Sixteen accessions of A. graveolens germplasm (Table 1) representing different countries of origin were selected. Seeds were obtained from the National Agrobiodiversity Center at the Rural Development Administration, Republic of Korea. RAPD analysis were performed using a single plant from each accession. DNA was extracted from freeze-dried leaves of 15-day-old seedlings from each accession, according to the modified CTAB method (Dellaporta et al., 1983). The relative purity and concentration of extracted DNA were estimated using a NanoDrop ND-1000 spectro-photometer (Dupont Agricultural Genomics Laboratory, NanoDrop Technologies, Wilmington, DE, USA). The final DNA concentration was adjusted to 10 ng/µl.

Table 1. List of Anethum graveolens accessions and details of collection sites.

Primer screening

 Three sets (OPA, OPB, and OPC) of 60 10-mer primers from Operon Technologies (Alameda, CA, USA) were ini-tially screened for their repeatable amplification with the 16 accessions. Primers were selected for further analysis based on their ability to detect distinct polymorphic amplified products across the accessions. To ensure reproducibility, the primers generating weak products were discarded.

PCR-RAPD

 The PCR-RAPD procedure for dill (A. graveolens) germ-plasm was carried out as described by Williams  et al.(1990). The amplification reaction was performed in a vol-ume of 25 µl containing 1× PCR buffer (10mM Tris–HCl, pH8.3, and 50 mM KCl) (NeoTherm; Gene Craft, Muenster, Germany), 0.2 mM dNTPs (NeoTherm), 3 mM MgCl₂, 1U Taq polymerase (NeoTherm), and 0.2 mM primer (Operon Technologies); then approximately 50 - 60 ng DNA tem-plate was amplified in a PTC-220 thermal cycler (MJ Research, Watertown, MA, USA). The following reaction conditions were used: initial denaturation for 4 min at 94°C, followed by 40 cycles of 1 min at 94°C (denaturation), 1 min at 36°C (annealing), and 2 min at 72°C (extension), with a final extension at 72°C for 10 min. The RAPD fragments were separated on a 1.5% agarose gel in 1× TAE (40 mM Tris–acetate, pH 8, 1 mM EDTA) buffer at 80 volt and 100 mA for 3 h. A  DNA (digested by PstI) ladder ranging from 150 bp to 20 kb was used as a molecular weight marker. The reproducibility of the amplification products was checked twice for each primer. A negative control that contained all components of a typical reaction but lacked template DNA was included on each gel. After electrophoresis, the gels were stained in ethidium bromide for 30min; thereafter, the gels were observed and photographed under ultraviolet light.

Data analysis

 The banding patterns obtained from RAPD were scored as present (1) or absent (0), each of which was treated as an inde-pendent character. Nei’s similarity matrix was subjected to clus-ter analysis by the unweighted pair group method for arithmetic mean averages (UPGMA) and a dendrogram was also gener-ated using the software MEGA 4. POPGENE 32 software was used to calculate Nei’s unbiased genetic distance among the dif-ferent genotypes for all markers. Data for Nei’s genetic diver-sity (H) and Shannon’s information index (I) across all 16 accessions were also analyzed (Zhao et al., 2006).

RESULTS

Marker polymorphisms

 The dill species were analyzed using three sets (OPA, OPB, and OPC) of 60 10-mer primers, of which 12 pro-duced reproducible polymorphic banding patterns (Fig. 1). In total, 119 bands were scored; 109 (91.6%) were polymor-phic. The number of bands generated per primer varied from 5 to 14 and a minimum of 5 bands was generated by the primer OPB 20, while a maximum of 14 bands was gener-ated by the primer OPC20. The size of the amplified prod-ucts varied from 200 to 3000 bp (Fig. 2). Seven primers(OPB07, OPB12, OPB13, OPB15, OPB18, OPB20, and OPC01) generated 100% polymorphic bands. A high level of polymorphism was detected by the RAPD markers in this study. Data for Nei’s genetic diversity (H) values ranged from 0.127 to 0.278 with a mean value of 0.214, while Shannon’s information index (I) ranged from 0.228 to 0.441 with a mean value of 0.353 (Table 2). The respective values of H and I were found to be highest for primer OPB15 and lowest for primer OPC04 in all 16 accessions and were ana-lyzed using 12 RAPD markers. The high values for Nei’s genetic diversity and Shannon’s information index indicated substantial variations within the population.

Fig. 1. RAPD primers (OPC05, OPC06, OPC07, OPC08, OPC09 and OPC10), amplification and screening of selected accessions of dill (Anethum graveolens).

Fig. 2. RAPD banding profile of 16 different dill accessions using primers A: OPC04 and B: OPC11; Lane-M, DNA marker, 1-16 Lane with primers (OPC04 and OPC11) represents RAPD profile.

Table 2. List of primers, number of scored bands, polymorphic bands, polymorphic percentages, Nei’s genetic diversity (H), Shannon information index (I), and standard deviation (S.D.).

Genetic diversity

 Jaccard’s genetic similarity coefficient varied from 0.00 to 0.64 (Table 3). The highest genetic similarity coefficient(0.64) was observed between  A. graveolens  accessions Unknown 1 and Georgia-2. UPGMA cluster analysis of the Jaccard’s similarity coefficient generated a dendrogram(Fig. 3). The dendrogram was developed for all 16 acces-sions of A. graveolens using RAPD markers. The genetic relationship among accessions using 12 RAPD markers, were grouped into three clusters, denoted as group-1, group-2, and group-3, and one out group. Group-1 contained 2 accessions (unknown1 and unknown2). Group-2 contained 11 accessions (Russia-1, Mongolia-1, Cuba, Georgia-2, Tajikistan, Nepal, Kazakhstan, Russia-2, North Korea, Mex-ico, and Mongolia-2). Group-3 contained 2 accessions(Ukraine-1, Uzbekistan-1) and the outgroup contained one accession (Georgia-1). Despite the identification of several groups, these dendrograms showed no strong relationships with respect to geographical distribution.

Table 3. Jaccard’s similarity coefficient for 16 Anethum graveolens accessions based on RAPD data analysis.

Fig. 3. UPGMA dendrogram showing the genetic relationships among the 16 Anethum graveolens accessions.

DISCUSSION

 Genetic diversity using molecular markers has been previ-ously described in A. graveolens  species (Jana & Shekha-wat, 2012) and AFLP was used by Solouki  et al. (2012). Study of genetic diversity in the family Apiaceae using molecular markers has been reported in Changium smyrnio-ides (RAPD) by Fu et al. (2003), Carum L. (internal tran-scribed spacers, ITS) by Papini  et al. (2007) and carrot(AFLP) by Santos & Simon (2002). Our study assessed the genetic diversity of 16 A. graveolens  accessions based on RAPD markers. Of the 60 random primers used, 12 gave reproducible amplification banding patterns for 109 poly-morphic bands out of 119 bands scored, accounting for 91.6% of the polymorphisms across all accessions. Seven primers (OPB07, OPB12, OPB13, OPB15, OPB18, OPB20, and OPC01) generated 100% polymorphic patterns.

Solouki  et al. (2012) reported a relatively low (39.8%) polymorphic percentage score in their research using AFLP in A. graveolens. In our RAPD analysis, significant genetic polymorphism was observed among the accessions of dill. These studies indicated that high genetic diversity may be attributable to geographic isolation, which has played an important role during the process of genetic diversification and variation (Guo et al., 2007). 

 In the present investigation, Nei’s genetic diversity (H) values ranged from 0.127 to 0.278 with a mean value of 0.214, while Shannon’s information index (I) ranged from 0.228 to 0.441 with a mean value of 0.353 (Table 2). The respective values of H and I were found to be highest for primer OPB15 and lowest for primer OPC04, in all 16 accessions analyzed using 12 RAPD markers. The high val-ues for Nei’s genetic diversity and Shannon’s information index indicated substantial variations within the population. In our study,  A. graveolens  maintained a high level of within-population genetic diversity and was a typical exam-ple of an outcrossing species (Snell & Aarssen, 2005). The genetic diversity of plant populations are largely influenced by factors such as the mating system, genetic drift, evolu-tionary history, and life history (Loveless & Hamrick, 1984). The present study corroborated the general concept of out-crossing species being characterized by their innate ability to possess higher levels of genetic diversity than selfing and clonal plants (Rossetto  et al., 1995). In the present study, Jaccard’s coefficient of similarity varied from 0.00 to 0.64, which is indicative of a high level of genetic variation among all the studied accessions. UPGMA cluster analysis indicated three clusters denoted as group-1, group-2, and group-3, and one outgroup. Despite the identification of sev-eral groups, the resulting dendrogram showed no strong relationship with respect to geographical distribution.

Solouki et al. (2012) also reported that their cluster analy-sis in A. graveolens  species showed that genetic diversity based on morphological traits was not related to geographi-cal distribution. These observations clearly indicated that the association between genetic similarity and geographical dis-tance was less significant. However, one must use a larger number of accessions from each geographical location to confirm the current pattern. The other factor of paramount importance is the influence of the environment and human activity over time (Solouki et al., 2008).

 In conclusion, the selection of RAPD markers for investi-gating genetic diversity in dill and the integration of infor-mation obtained by molecular analysis will allow the formulation of effective conservation strategies. In this study, the limited number of populations tested does not rep-resent the whole genetic variability in A. graveolens. A need appears to exist for maintaining sufficiently large popula-tions for conservation of its genetic diversity and avoiding genetic erosion.

ACKNOWLEDGEMENT

 This study was carried out with the support of “Research Program for Agricultural Science & Technology Develop-ment (Project No. PJ008623)”, National Academy of Agri-cultural Science, Rural Development Administration, Republic of Korea.