PHYLOGENETIC ANALYSIS OF DIFFERENT ARTEMISIA SPECIES BASED ON CHLOROPLAST GENE RPS11

Artemisia L. of the family Astereacae is a genus with enormous economical and medicinal importance. As a result, the genus Artemisia has been the subject of diversity-focused studies. In the present study, phylogenetic analysis based on restriction fragment length polymorphism of the chloroplast rps11 gene was conducted on eight species of Artemisia that represent the major morphological subgroups. After digestion of the rps11 gene that was amplified from eight species, with six different restriction enzymes, each restriction site was observed and scored on a 12% polyacrylamide gel. The data were analyzed using the Numerical Taxonomy and Multivariate Analysis System to infer the phylogenetic relationship within the genus. A mixed pattern was observed among the species belonging to various taxonomic groups of Artemisia.


INTRODUCTION
Artemisia is considered to be the largest genus present in the tribe Anthemideae and it is the largest genera in the family Asteraceae, having more than 500 taxa (the number varies depending on different reports: McArthur, 1978;Ling, 1982;Mabberley, 1990;Bremer and Humphires, 1993;Oberprieler, 2001;Valles and McArthur, 2001).Artemisia is widely distributed in the northern hemisphere with two of its main speciation centers present in western and central Asia and a few of its representatives present in the southern hemisphere.Pakistan is considered to be one of the centers of origin of Artemisia species and many species of Artemisia have been reported (Pareto, 1985;Tan et al., 1998).After different systematic reshufflings, the genus was divided into five large groups; Absinthium DC., Artemisia L., Dracunculus Besser, Seriphidium Besser and Tridantatae (Rydb.)(Torrell et al., 1999).
The genus Artemisia is an important medicinal plant and also has high economical value with respect to its usage as food, forage and ornamental plants ( Barney and DiTommase, 2003).It has also been reported that in many countries of the Middle East and Turkey many species of Artemisia are used as herbal medicines for the treatment of diabetes, high blood pressure and gastrointestinal ailments (Mossa, 1985;Al-Shamaony et al., 1994;Subramoniam et al., 1996).Artemisia species such as A. annua and A. indica contain a drug called artemisinin that has been used for treating malaria in China and Thailand for over 2000 years (Bunyapraphatsara, 1986;Farnsworth, 1992;Ashraf et al., 2010).A. dubia has been used in Magar of Bukini, Baglung, Western Nepal as a stomachic and purgative, and for asthma and skin diseases such as scabies and ulcers (Sapkota, 2008).A. absinthium L. is known to possess ethno-medical and biological properties related to anthelmintic (Tariq et al., 2009), antifungal (Kordali et al., 2005) and antimicrobial activities (Lopes-Lutz et al., 2008).Moreover, Afsanteen (A. brevifolia) is widely used in the ethno-veterinary medicine system of Pakistan as an anthelmintic plant (Iqbal et al., 2004).
Different studies have been conducted to measure the genetic diversity or phylogenetic analysis of Artemisia species by using different molecular markers (Mohsen and Ali, 2008;Hayat et al., 2009;Nazar and Mahmood, 2010).Besides the other molecular markers related to techniques for phylogenetic studies, cpDNA is a genome frequently used in population genetic studies (Comes and Abbott, 1999), and it has successfully been used for phylogenetic analysis (Ziegenhagen and Fladung, 1997).Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) has the ability to discriminate between genotypes based upon the presence or absence of restriction sites within the amplified DNA.This technique is also called Cleaved Amplified Polymorphic Sequence (CAPS) (Karp et al., 1997).In the present study, the phylogenetic relationship amongst eight Artemisia species has been carried out with the help of a PCR-RFLP-based analysis of rps 11, a gene encoding chloroplast ribosomal protein 11.

Collection of plant material
Artemisia samples were collected from different areas of Pakistan (Table 1).

Primer designing and amplification of the rps11 gene
A pair of primers was designed from tobacco chloroplast genome (Accession # Z00044.2) available in Genbank for the amplification of ribosomal protein of smaller subunit 11 (rps 11).Primers were designed with the help of online program Primer 3. The sequence of primers is as follows; rps11F: 5' TGGCAAAAGCTATACCGAAAA 3' rps11R: 5' TTCGGAGGTCTACAGCCATT 3' The PCR conditions used for amplification of the rps11 gene were pre-PCR denaturation at 94˚C for 5 min followed by 35 cycles of denaturation at 94˚C for 30 s, annealing at 55˚C for 30 s and extension at 72˚C for 1 min.Final extension was carried out at 72˚C for 20 min and the PCR reaction contents were held at 4˚C till further use.About 25 µl of the PCR mixture contained, 12.5 µl of PCR Master Mix (Fermentas), 9.5 µl PCR water, 1 µl of each primer (25 pM) and 1 µl of DNA template was used.

Cleaved Amplified Polymorphic Sequence (CAPS)
Restriction enzymes were selected by digesting the already available sequence of rps11 gene using the online tool NEB cutter (http://tools.neb.com/NEBcutter).Then, the selected restriction enzymes were used for the digestion of the rps11 gene amplified from eight Artemisia species.The digestion mixture (containing the PCR amplified product (10µl), nuclease free water (7 µl), 10X Buffer (2µl), and enzyme (5 units; 1µl) (Fermentas)) was incubated overnight.After incubation, the digested product was subjected to 12% polyacrylamide gel (BioRad) electrophoresis and then silver stained.The photographs of the gels were taken with a SONY Cyber-Shot 10.1 mega pixel camera.

DATA SCORING AND ANALYSIS
The presence of a particular restricted fragment was marked as "1" and its absence as "0".For phyloge-

RESULTS AND DISCUSSION
After various taxonomic studies the genus Artemisia was divided into five large groups; Absinthium DC., Artemisia (=Abrotanum Besser), Dracunculus Besser, Seriphidium Besser and Tridantatae (Rydb.)(Torrell et al., 1999).However, this infrageneric classification does not represent natural groups and is not accepted by all taxonomists (Persson, 1974;McArthur et al., 1981;Valles and McArthur, 2001;Valles and Garnatje, 2005).Through various molecular studies (Sanz et al., 2008;Tkach et al., 2007;Valles et al., 2003;Watson et al., 2002) it was concluded that the genus may need more molecular evidence to resolve the confusions at the infrageneric level.In the present phylogenetic analysis an attempt has been made to find out the relationships among different species of genus Artemisia by using the PCR-RFLP technique.
Eight Artemisia species were collected from different parts of Pakistan and the plants were preserved at -20˚C.High quality DNA was isolated from the leaves of preserved plants by the CTAB (cetyl trimethyl ammonium bromide) isolation procedure (Richard, 1997).The isolated DNA was used for amplification of the rps11 gene.The amplified product of approximately 400 bp was subjected to restriction digestion with six different restriction enzymes (ScrfI, TscAI, DpnI, HinfI, BsiHKAI and FokI).The digested product was run on 12% PAGE.In total, 77 restriction fragments were observed: the highest number of fragments was produced by DpnI (16 fragments), and the lowest by BsiHkAI (9 fragments).It was also observed that the amplified rps11 gene remained un-  digested in some species probably due to site variations (Table 2).

Phylogenetic analysis
The data obtained from the RFLP of the rps11 gene was used to reveal the phylogenetic relationship among the species of Artemisia.UPGMA cluster diverge at 38% similarity coefficient into two lines.
All the species grouped into one cluster, except A. vulgaris which was parallel to all the other species.Earlier, Barney and DiTommase (2003) reported a high level of intraspecific diversity in A. vulgaris in comparison to other species of Artemisia.Moreover, A. japonica and A. persica of sections Dracunculus Besser and Absinthium DC, respectively, appeared at 100% similarity, thus revealing a very close association during the evolutionary process.Furthermore, both species showed a close link with A. brevifolia (belonging to section Tridantatae (Rydb.)McArthur).It was observed that these three species formed a group and they showed a relationship with A. tangutica and A. roxburghiana at 74% similarity coefficient.Similarly, A. tournifortiana and A. dubia diverged at 68% similarity coefficient.
It is evident from the data that mixing had occurred at an infrageneric level during the evolutionary process.Sometimes geological and ecological factors also affect the genetic characterization and organiza-tion of diversity (Loveless and Hamrick, 1984).To resolve the problem of genus Artemisia at an infrageneric level there is a need to study other genomic regions to produce groups depicting natural classification.

Table 1 :
List of Artemisia species collected from different regions of Pakistan.

Table 2 :
Total number of restricted fragments produced by different restriction enzymes among eight species of Artemisia.
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