DNA Barcoding of Ricinus communis from Different Geographical Origin by Using Chloroplast matK and Internal Transcribed Spacers

Ricinus communis have attracted considerable attention because of its specific industrial and pharmacological activities. DNA barcodes can be used as reliable tools to facilitate the identification of medicinal plants for the safe use, quality control and forensic investigation. In this study, the differential identification of eight accessions of R. communis was investigated through DNA sequence analysis of two candidate DNA barcodes. The nucleotide sequence of internal transcribed spacers (ITS2) and chloroplast maturase gene (matK) have been determined to construct the phylogenetic tree. The phylogenetic relationships of accessions based on the nrITS2 region and partial matK region showed that all accessions in this study were related to three geographical origins. Based on sequence alignment and phylogenetic analyses we concluded that the ITS2 sequences can distinguish R. communis accessions from different geographical distributions.


Introduction
The castor oil plant, Ricinus communis also known as Palma(e) christi or wonder tree.It is a perennial scrub of the family Euphorbiaceae.The plant can vary greatly in its growth habit and morphology.The variability has been increased by breeders who have selected a range of cultivars for use as ornamental plants, and for commercial production of castor oil [1].The castor oil is a wonderful universal remedy for a large number of health concerns.The oil has been used as for warts, cold tumors, indurations of the abdominal organs, lacteal tumors and indurations of the mammary gland [2].Seeds have high oil content, with multiple industrial applications such as paints, lubricants, cosmetics, polymers and biofuels [3].DNA barcoding is a method of describing and identifying species by analyzing sequence information from one or a few short standardized loci amplified with universal primers.To standardize the international use of DNA barcodes, the scientific community has made considerable efforts searching for suitable DNA regions to barcode every species [4][5][6][7][8].DNA barcoding provides a rapid identification tool, utilizing only minute amount of tissue from any stage of development of a plant or animal.DNA barcodes can be used to identify specimens correctly, to expand the discovery of new species, in tackling illegal trade of endangered species of both plant and animals and in forensic investigation to help detect poisonous materials in life-threatening cases [9,10].DNA barcoding has been proposed as a novel and powerful taxonomic tool [6,11], the mitochondrial cytochrome oxidase subunit I (CO1) is a widely used barcode in a range of animal groups [12][13][14][15] this locus is unsuitable for use in plants due to its low mutation rate [4,14,15].A variety of loci have been suggested as DNA barcodes for plants, including coding genes in plastid genome and the multicopy nuclear Internal Transcribed Spacer (ITS) are two of the leading candidates [4].Thus, this issue is addressed in the present study by comparing the feasibility of using each of these proposed DNA barcodes (matK, ITS1, ITS2) to identify genetic variations of R. communis accessions from different geographical distributions.

DNA Isolation
Plant seeds are hard to lyse due to seed coat and hard cotyledon inside, the seed coats mainly contain tannins that inhibit PCR.The inner seed material contains starches and lipids that can foam and make lysis difficult [16].The seeds of each accession were immersed in liquid nitrogen and crushed using sterile mortar and pestle to get a fine powder.An automatic DNA extraction (Maxwell 16 , Promega) and DNeasy plant mini kit (Qiagen) were used for DNA extraction.DNeasy plant mini kit with slight modification in which 0.4% (v/v) β-mercaptoethanol was added to AP1/E lysis buffer was performed.Quality of the extracted DNA was determined using gel electrophoresis.

PCR Amplification
A total volume of 30 µL of PCR reaction mixture contained the following: 15 µL of PCR Master Mix (Qiagen, Germany), giving a final concentration of 200 mM each deoxynucleotide and 1.5 mM MgCl 2 , 20 pM each primer (Table 2), 2 µL of genomic DNA (50 ng) and the rest was adjusted with sterile distilled water.PCR amplification was performed with a thermal cycler (T 100 , BIORAD) as follows: one cycle at 95˚C for 5 min, followed by 35 cycles of 95˚C for 30 s, 55˚C for 30 s and 72˚C for 1 min, followed by an elongation step at 72˚C for 5 min.All the PCR conditions were the same for all the primer pairs.

Agarose Gel Electrophoresis
PCR products were examined using 1.5% agarose gel electrophoresis in 1X TBE (Tris-Boric acid-EDTA) buffer at 70 V for ∼45 min.Gel images were obtained using Gel documentation (Major Bioscience, Taiwan) imaging system.The size of PCR products resulting from the primer pairs of the specific barcoding gene were determined by using a 50 bp sharp mass (Euro Clone, Italy) and 100 bp DNA ladder (Promega, Madison).

Sequencing and Alignment
PCR products sent to the Source Bioscience (Nottingham, UK) for sequencing after simple purification and sequenced according to the method originally described by Sanger [17].ITS2 and matK products were sequenced using the same primer pairs as used for the initial amplification.The sequences from each DNA region were aligned by CLUSTALW and genetic distance was computed using the MEGA 5.0 Kimura two-parameter (K2P) model [18].The nucleotide sequence data of the partial matK sequence and ITS2 spacer were deposited in the Genbank nucleotide sequence databases with the accession numbers reported in Table 2.The phylogenetic trees were constructed using maximum likelihood (ML) in MEGA 5.0 software program.Bootstrap testing of 1000 replicates was performed to estimate the confidence level of the topology of the consensus tree.

Results
Our study showed that DNA extraction using Plant DNeasy minikit provided better yield and quality compared with automatic DNA extraction method that failed to produce higher quality and PCR amplification.For the ITS and matK, all samples showed an equal size of the PCR product.Excluding the primer flanking sites, the sizes of the ITS1, ITS2 and matK of all accessions were 360 to 440 and 790 bp in length, respectively as shown in Figure 1.The ITS1, ITS2 spacers and matK gene of all accessions were successfully amplified and only ITS2 and matK regions were successfully sequenced.For a     3).In contrast, matK sequence divergence among 8 accessions varied was from 0.13 % to 0.75% (Table 4).The phylogenetic tree constructed by the matK gene analysis suggested that the eight accessions were divided into two clusters.R2 (Egypt) belong to the same cluster with R1 (Kadiogo), R5 (Tunisia), R6 (UAE), R7 (Venezuela), R8 (Hadramout), and R9 (Lawdar), while R3 (Lebanon, Sour) separated into another cluster (Figure 4).This tree was incompatible with that constructed by ITS2 analysis suggested that R7 (Venezuela) and R2 (Egypt), and R1 (Kadiogo) were in one cluster, other accessions (R5, R8, R9, R6 and R3) in the second cluster which were divided into two subclusters in the phylogenetic tree (Figure 5).

Discussion
The castor oil plant R. communis is one of the oldest drugs known to man.The first mention of it as a laxative can be found in 3500 year-old Ancient Egyptian papyrus scrolls.The most promising plastid candidate maturase K [15,19] was tested, along with the nuclear locus internal transcribed spacer (ITS2), which is also a most important candidate for plant barcoding [4,20].The ITS2 region was selected as a barcode candidate because ITS2 sequences are potential general phylogenetic markers and are widely used for phylogenetic constructions at both the genus and species levels [21,22].As the ITS2 region is one of the most common regions used for phylogenetic analyses [23].In our study, nrITS1 regions were amplified cleanly in 8 accessions but sequencing was unsuccessful.Chodon et al. [24] reported that one of potentially negative factor for sequencing nrITS is the presence of ply-G, poly-C, and poly-A repeats.In general the nrITS2 region is more length-conserved than nrITS1, making it a more predictable amplicon to work with [7].
Our research shows that a single region matK or ITS2 a portion, it was demonstrated that the sequence nucleotide variation can distinguish genetics divergence among R. communis from different geographical origin; this was supported by sequence alignment analyses.In previous studies, ITS2 has already been suggested as a suitable marker applicable for phylogenetic reconstruction in eukaryotes by many researchers [21,22,25].The matK coding region is one of the most rapidly evolving regions in chloroplasts and shows a high level of species discrimination among angiosperms, a fragment of 600 -800 bp is    usually sufficient [15,26].The matK region varied sufficiently to distinguish "Sanqi" (Panax notoginseng; Araliaceae) from different geographical origins [27], but it failed to differentiate among Sanqi cultivars [28].The partial matK sequence of 7 accessions (R1, R5, R6, R7, R8, and R9) shows only one nucleotide substations at  one position compared with accession R2 from Egypt.However, R3 accession from Lebanon had 16 base substitutions.In this study accessions from Yemen, Kadiogo, Venezuela, United Arab Emirates and Tunisia had the same matK sequence which might be ascribed to the same ancestor and different Environment.In the present study, nrITS2 sequence was found to correlate with geographical of the samples which matK gene sequence was conserved than the ITS2.

Conclusion
Based on our own findings, we propose that ITS2 be used as the desired barcode to study geographical distributions of Euphorbiaceae species.

Figure 2 .
Figure 2. Part aligned sequence of the ITS2 region of eight accessions of R. communis.The dots indicate that the base at that position in the specified sequence is the same as the base in the sequence written at the top of the compilation.

Figure 3 .Figure 4 .
Figure 3. Part aligned sequence of the matK barcode region of eight accessions of R. communis.The dots indicate that the base at that position in the specified sequence is the same as the base in the sequence written at the top of the compilation.matKR8matKR7 matKR6 matKR5 matKR1 matKR9 matKR2 matKR3 Figure 4. Maximum likelihood tree constructed by partial sequence of matK gene from eight R. communis accession.

Table 1 . Plant samples used in this study.
*The number is the serial number in the Millennium Seed Bank (MBS), Kew Royal Botanic Gardens.