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Genetic variation among sumac (Rhus coriaria L.) samples collected from three locations in Jordan as revealed by Aflp markers.

Introduction

Rhus coriaria commonly called sumac or sumach, is a perennial plant that belongs to the anacardiaceae family, one of approximately 250 species of the Rhus genus. This species is widely distributed in uplands with elevation (1200m a.s.l) mainly in Ajloun, Irbid and Jerash areas in Jordan. It is characterized by deciduous spirally arranged leaves, pinnately compound, densely panicle or spike flowers with red color and dense clusters of reddish drupes fruits. It is propagated by new sprouts from rhizomes or seeds (Sumac. http:// en.wikipedia.org/ wiki/sumac).

Rhus coriaria species was planted at the roadsides, ornamental and forest protected areas to conserve the soil from erosion. Moreover, it was used in folk medicine to alleviate stomach problems, diarrhea, bleeding and skin problems [12].

In Turkey and other Arabic countries, the sumac berry was used as a cooking spice (http:// unitproj.library.ucla.edu/bioned/spice/index.cfm); in addition it is considered an important ingredient of thyme (Zatter) and sesame mix with olive oil which is desired by many people with breakfast dishes. Ozcan and Haciseferogullari [12] reported that sumac is a very popular condiment used as a major souring agent, mixed with freshly cut onions and considered as appetizer. Jordanian people collected the fruits and flowers and processed them to become edible and then marketed to improve their income.

However, the water extract of sumac Rhus coriaria has a positive effect on the positive gram bacteria [9,10], also its extract was shown to inhibit the formation of hydroperoxide in natural peanut oil stored at 65[degrees]C for 35 days [2].

Genetic diversity has been studied based on the morphological, physiological and chemical analysis. Goulao et al. [6], reported that the traditional methods for characterization and assessment of genetic variability based on morphological, physiological and agronomic traits are often not adequate.

However, recently molecular analys is solved the problem in a few hours compared to months or years. Many molecular markers techniques such as Amplified Fragment Length Polymorphism AFLP, were used to distinguish between individuals, species, accessions and varieties. AFLP markers have been developed by Vos et al., [16] and widely spread due to its reliability and robustness. This technique based on four steps restriction/ligation, pre-selective, selective and gel electrophoresis.

The use of molecular markers is speeding up plant breeding and clarifying, confirming or even reformulating the systematic taxonomy of several groups of organisms [15]. AFLP markers complement the traditional morphological and phonological descriptors used for the registration of new cultivars. They also contribute to the protection of intellectual property and allow the certification of clonally propagated varieties [4]. The AFLP method [16] has been widely employed in research of plants, fungi and bacteria [1]. The AFLP technique have been used to study the genetic relationships among Mediterranean pistacia species [5], apple cultivars [6], Miscanthus [7], Brassica nigra accessions [11] and European Rubus [14]. In addition, intras pecific diversity reported in woody plants using AFLP markers is very high such as in olive [13].

The aim of this study was to examine the genetic relationship among sumac samples that were collected from different sites in Jordan based on AFLP markers.

Materials and methods

Plant material

Leaves were collected randomly from 10 sumac trees from Jordan namely, Irbid, Ajloun and Jerash (Table 1).

DNA isolation

Total cellular DNA was extracted using a modified CTAB isolation protocol. Approximately (30 mg) fresh leaves was ground in liquid nitrogen and mixed with 750 [micro]l of fresh and preheated 2x CTAB solution with 0.8g PVPP in 2ml tubes then placed at 65[degrees]C for 30 min. The mixture was mixed with 750 [micro]l of chlorophorm/isoamyl alcohol (24:1) then vortexed a few seconds, then centrifuged at 14000g for 20 min. The upper phase (supernatant) was placed in 2ml tubes with 600ml isopropanol and the tubes were then shaked until the thread of DNA appeared before they were centrifuged for 20 min at 14000g. The solution was poured off and the tubes left to dry before adding 600 [micro]l of cold 70% ethanol and then placed overnight at -20[degrees]C. The ethanol was then poured off and the tubes dried. 100 [micro]l of TE was added and placed at 65[degrees]C for 30min. Four micolitter of RNAse (10mg/ml) were added per tube and left for 45min at 37[degrees]C. DNA quantitation was performed using a S2100 UV/VIS DIODE-ArraySpectrophotometer, Version 1.7.

AFLP procedure

The AFLP procedure was performed as previously described by Vose et al. [16]. Sumac DNA (500ng) was double digested with EcoRI and Tru9' (an isochizomer of Msel) and double stranded adaptors were ligated to the ends of DNA fragments, generating template DNA for subsequent PCR amplification (pre-amplification followed by selective). Restriction and ligation reactions were carried out simultaneously in a single reaction tube [16].

To carry o ut the reaction, an enzyme master mix for 35 reactions was prepared containing 2[micro]l EcoRI buffer, 2[micro]l Tru9' buffer, 0.25[micro]l EcoRI enzyme (12[micro]/[micro]l), 0.25[micro]l Tru9' enzyme (12[micro]/[micro]l), 0.625[micro]l 1mg/ml BSA, 9.875[micro]l ddH2O and 5[micro]l genomic DNA (500ng), the mix was centrifuged for a few seconds then stored at 37[degrees]C for 3 hr. The restriction-ligation reactions consisted of 0.5[micro]l 10x T4 buffer, 0.2[micro]l T4 DNA ligase (3[micro]/[micro]l), 0.5[micro]l Tru9' adaptor forward, 0.5[micro]l Tru9' adaptor reverse, 0.5[micro]l EcoRI adaptor forward, 0.5[micro]l EcoRI adaptor reverse and 2.3[micro]l ddH2O. Then distributed 5[micro]l for the restriction tubes, then stored at 37[degrees]C for 3hr. The restriction-ligation reactions were diluted 1:4, then used in preamplification step. The Tru9' complementary primer had a 3'-C and the EcoRI complementary primer had a 3'-A.

The pre-amplification (preselective) mix was prepared by adding 2[micro]l of 5-fold diluted DNA from the restriction-ligation reaction, 2.5[micro]l 10xbuffer (PCR), 2.5[micro]l dNTPs (5mM), 1[micro]l AFLP preselective primer (EcoRI+ A-3'), 1 [micro]l AFLP preselective primer (Tru91 + C-3'), 0.2[micro]l Taq polymerase (5[micro]/[micro]l) and 15.8[micro]l ddH2O. The pre-amplification (preselective) amplification was carried out in a thermal cycler programmed at 72[degrees]C for 2min followed by 20 cycles of 94[degrees]C for 30 sec, 56[degrees]C for 40 sec and 72[degrees]C for 50 sec and finally incubated at 4[degrees]C. The preamplification DNA was diluted 5-fold with ddH2O and selective amplifications were carried out by using different EcoRI and Tru91 primer combinations (Alpha DNA).

Primers selected for the selection amplification were from available AFLP selective primers that were purchased and stored at -20[degrees]C at NCARE lab. The EcoRI primers contained three selective nucleotides with sequence (Table 1) while the Tru91 primers had the selective nucleotides starting with G (Table 1).

For the selective amplification, the reactions were set up as follows: 2[micro]l of 5-fold diluted preselective amplification reaction product, 1.0[micro]l Tru91 primers, 1.0[micro]l EcoRI primers, 2[micro]l of 10 x buffer, 2[micro]l dNTPs (0.25mM), 0.2[micro]lTaq polymerase (5[micro]/[micro]l), 0.25[micro]l of MgCl2 (15mM) and 11.55ddH2O.

Selective amplification was carried out in a thermal cycler programmed at 94[degrees]C for 2min, followed by 13 cycles of 94[degrees]C for 20 sec, 68[degrees]C for 30 sec and 72[degrees]C for 1min, and 23 cycles of 94[degrees]C for 30 sec, 59[degrees]C for 30 sec and 72[degrees]C for 1min and a final incubation at 4[degrees]C. The selective amplification reaction product (6[micro]l) was mixed with 4[micro]l of loading buffer (98% deionized foramide, 10mM EDTA (pH: 8), 0.05% bromofenol, 0.05% xylene cyanol), from which 6[micro]l was finally loaded onto a 6% polyacrylamide gel run on a vertical gel sequencing apparatus (Cleaver, Scientific, Ltd.).

Nine primer combinations (EcoRI- / Tru91-) were used in analysis (Table 1), thirty samples from sumac trees were subjected to the selective amplification with these primer combinations, and treated under the same conditions. To determine the size of the AFLP fragments, we used an AFLP DNA marker (50bp step ladder) (Promega), ranging in length from 50 to 800bp.

Data analysis

AFLP polymorphic bands were scored as present (1) or absent (0) and estimates of similarity among all tested samples were calculated according to Ne and Li, (1979). The matrix of similarity was analyzed by the Unweighted Pair-Group Method (UPGMA) and the dendrogram was obtained by using SPSS program, version 10.

Results and Discussion

Out of nine primer pair combinations that were tes ted only three pair combinations ATA/CAA, AAG/CAG and AAT/CAT showed amplified fragments (Table 2). A high number of amplified fragments 25 and 17 were detected by the combinations ATA/CAA and AAG/CAG, respectively (Table 3). The primer pair combination AAT/CAT showed the lowest number of bands. The similarity ranged from 0.12 % to 1.00 % (Table 2). A high similarity index (1.00) was showed among the samples that were collected from Jerash governorate. These samples indicate that these sumac trees probably came from the same source of seeds and nursery of Jerash forest, which propagated them and then distributed to the farmers in the same region, but the lowes t similarity (0.12 %) registered between samples of Irbid and Irbid-Ajloun (Table 2).

However, the AFLP analysis show that their is a genetic variation among the sumac trees studied. This judgment based on the dendrogram (Fig 1) was generated among samples. Four main clusters were generated from this dendrogram.

[FIGURE 1 OMITTED]

The first group includes three subgroups, the first one formed 4 samples were more closely and the second subgroup formed two samples from Jerash the third showed one individual of Irbid. The second group formed two individuals from Irbid. The third group has three subgroups the first has one sample from Ajloun and one from Jerash, the second group included two samples from jerash but the final sub group has one Ajloun individual. The fourth group included three sub clusters the first has four sub- sub cluster, the second sub cluster has three sub-sub clusters but the rest sub cluster has two sub-sub cluster.

High genetic diversity was found among the three populations collected from three locations (Figure 1 and Table 2). For Jerash region, with the exception of the pair of samples 25, 24, 28 and 27, all other entries are genetically distinct but they formed two separated groups, the first included 7 individuals, the second included 3 individuals. For Irbid region, two individuals formed one group, one individual included with the first group, and seven individuals were grouped with Ajloun samples. Ajloun region relatively showed varied individuals through its formed two samples with Jerash and eight samples with Irbid in distinct groups. High similarity was showed between 17and 18 samples (Figure 1 and Table 2).

Irbid population has shown the highest intra population diversity since 7 sub-clusters can be visualized; five of them included one individual each. High similarity was showed between one sample from Ajlo un (20) and Jerash (23). The accessions 76 and 18 are the closest. High genetic diversity within a population was found. The similarity matrix allowed to confirm the high relatedness between the samples in the pairs (24, 25, 26, 27 and 28) of Jerash and (17 and 18) of Ajloun. The variability that was found may be related to the ecogeographical condition which plays a great role for creating variation through climatic changes over the past years.

Further study should be conducted to survey all of sumac species grown in Jordan and extend the research to include the studying of the chemicals (for example, Malic acid, citric acid and tartaric acid) and the physical properties. More efficient results will be obtained if all findings correlated with ecogeographical information and if it is possible to compare them with other s pecies grown within neighbor countries of Jordan such as Syria and Turkey etc. Knowledge of the genetic structure of a landrace is fundamental in elaborating strategies which involve the local farmers, allowing us to improve and safeguard the genetic integrity of landrace genetic resources [8]. AFLP markers enable a quick and reliable assessment of intraspecific genetic variability [3].

In conclusion, AFLP is a useful tool that can facilitate the collection and evaluation accessions or genetic resources and shortening the time needed for assess the genetic variation among and within individuals and species in the fields.

Acknowledgements

The authors are grateful to Dr. Abdel Nabi Fardous the previous of general director of NCARTT for his support and encouragement of the scientific research.

References

[1.] Bensch, S., K. Kesson, 2005. Ten years of AFLP in ecology and evolution: why so few animals. Molecular Ecology. 14: 2899-2914.

[2.] Dogan, M., A. Akgul, 2005. Characteristics and fatty acids compositions of Rhus coriaria cultivars from South East Turkey. Chemistry of Natural Compounds. 41: 724- 725.

[3.] Gaudeul, M., P. Taberlet, I. Till-Bottraud, 2000. Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers. Molecular Ecology, 9: 1625-1637.

[4.] Geuna, F., M. Toschi, D. Bassi, 2003. The use of AFLP markers for cultivar identification in apricot. Plant Breeding, 122: 526-531.

[5.] Golan-Goldhirsh, A., O. Barazani1, Z.S. Wang, D.K. Khadka, J.A. Saunders, V. Kostiukovsky, L.J. Rowland, 2004. Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers. Plant Syst. Evol.. 246: 9-18.

[6.] Goulao, L., L. Cabrita, C.M. Oliveira, J.M. Leitao, 2001. Comparing RAPD and AFLP analysis in discrimination and estimation of genetic similarities among apple (Malus domestica Borkh.) cultivars RAPD and AFLP analysis of apples. Euphytica, 119: 259-270.

[7.] Hodkinson, T., M.W. Chase, S.A. Renvoize, 2002. Characterization of a genetic resource collection for Miscanthus (Saccharinae, Andropogoneae, Poaceae) using AFLP and ISSR PCR. Annuals of Botany, 89: 627-630.

[8.] Lioi, L., A.R. Piergiovanni, D. Pignone, S. Puglisi, M. Santantonio, G. Sonnante, 2005. Genetic diversity of some surviving on-farm Italian common bean (Phaseolus vulgaris L.) Landraces. Plant Breeding, 124: 576-581.

[9.] Naser-Abbas, S.M., A.K. Halkman, 2004. Antimicrobial effect of water extract of sumac (Rhus coriaria L.) on the growth of some food borne bacteria including pathogens. Int. J. Food. Microbiol., 97: 63-69.

[10.] Naser-Abbas, S.M., Kadir A. Halkman, M.I. Al Haq, 2004. Inhibition of some food borne bacteria by alcohol extract of sumac (Rhus coriaria L.). J. Food Safety, 24: 257-267.

[11.] Nei, M., W.H. Li, 1979. Mathematical model for s tudying genetic variation in terns of restriction endonucleases. Proc. Natl. Acad. Soc., 76: 5269-5273.

[12.] Ozcan, M., H. Haciseferogullari, 2004. A condiment sumac (Rhus coriaria L.) fruits: some physioco-chemical properties. Bulg. J. Plant Physiol., 30: 74-84.

[13.] Sanz-Cortes, F., D.E. Parfitt, C. Romero, D. Struss, Lla' G. cer, M.L. Badenes, 2003. Intraspecific olive diversity assessed with AFLP. Plant Breeding, 122: 173-177.

[14.] Steinger, T., B.A. Roy, J. Kollmann, 2002. Evidence of sexuality in European Rubus (Rosaceae) species based on AFLP and allozyme analysis. American J. Botany, 87: 1592-1598.

[15.] Talhinhas, P., J. Neves-Martins, J. Leitao, 2003. AFLP, ISSR and RAPD markers reveal high levels of genetic diversity among Lupinus spp. Plant Breeding, 122: 507-510.

[16.] Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. Van de lee, M. Hornes, A. Fijters, J. Pot, J. Peleman, M. Kuiper, M. Zabeau, 1995. AFLP: Anew technique for DNA fingerprinting. Nucleic acid Res., 23: 4404- 4414.

(1) Ibrahim Mohammad Rawashdeh, (2) Abudel Latif Ghzawi, (1) Nasab Q. Rawashdeh, (3) Kamal Khairallh, (4) Abdel Rahman Al-Tawaha and (3) Bannur Salama.

(1) Medicinal plants and biodiversity program, National Center for Agricultural Research and extention (NCARE), P. O. Box 639. Baq'a, Jordan.

(2) Biotechnologists, Hashimiah University.

(3) Manager GEF/WB project conservation of medicinal and herbal plants of Jordan.

(3) Bannur Salama, Biotechnology research centers, Tripoli, Libya. Banor2004@ yahoo.com

(4) Department of Biological Sciences, Al Hussein Bin Talal University Ma'an, P.O. Box 20, Jordan.,

Corresponding Author Ibrahim Mohammad Rawashdeh, Medicinal plants and biodiversity program, National Center for Agricultural Research and extention (NCARE), P. O. Box 639. Baq'a, Jordan. E-mail: irawashdeh2002@yahoo.com.
Table 1: Oligonucleotides adaptors and primer
combinations used for AFLP analysis.

Name                        Sequence

EcoRI adaptor               5'- CTCGTAGACTGCGTACC-3'
                            3'- AATTGGTACGCAGTC-5'
  Tru91 adaptor             5'-GACGATAGTCCTGAG-3'
                            3'-TACTCAGGACTCAT-5'

Primers used in
pre-amplification
EcoRI+ 1-A                  5'-GACTGCGTACCAATTCA-3'
Tru91 + 1-C                 5'-GAT GAGT CCT GAGTAAC -3'

Primers used in
selective amplification
EcoRI+ 3-ACA                5'-GACTGCGTACCAATTC+ACA-3'
EcoRI+ 3-AAG                5'-GACTGCGTACCAATTC+AAG-3'
EcoRI+3-AAC                 5'-GACTGCGTACCAATTC+ATA-3'
EcoRI+3-ATA                 5'-GACTGCGTACCAATTC+ATA-3'
EcoRI+3-AAT                 5'-GACTGCGTACCAATTC+AAT-3'

MseI (an isochizomer
of Tru91)
MseI+3-CAT                  5'-GATGAGTCCTGAGTAAC+CAT -3'
MseI+3-CAG                  5'-GAT GAGT CCT GAGTAAC+CAG-3'
MseI+3-CAA                  5'-GAT GAGT CCT GAGTAAC+CAA-3'

Primer pair combinations
(EcoRI/ MseI).
1-ACA/CAT
2-AAG/CAG
3-AAG/CAT
4-AAC/CAT
5-AAC/CAG
6-ATA/CAA
7-AAT/CAT

Table 2: Similarity matrix based on the AFLP markers
among thirty trees of sumac collected from three
locations in Jordan during 2007.

       1      2      3      4      5      6      7       8     9

1
2    0.40
3    0.24   0.38
4    0.18   0.40   0.50
5    0.17   0.33   0.38   0.75
6    0.34   0.43   0.33   0.27   0.43
7    0.20   0.27   0.27   0.29   0.27   0.31
8    0.29   0.19   0.19   0.29   0.27   0.21   0.46
9    0.20   0.12   0.19   0.13   0.19   0.21   0.14   0.14
10   0.20   0.27   0.36   0.39   0.27   0.31   0.23   0.23   0.23
11   0.18   0.24   0.31   0.13   0.24   0.15   0.20   0.13   0.20
12   0.18   0.24   0.24   0.13   0.24   0.27   0.29   0.20   0.20
13   0.27   0.33   0.25   0.14   0.25   0.50   0.21   0.13   0.21
14   0.12   0.25   0.25   0.14   0.25   0.29   0.21   0.13   0.21
15   0.13   0.19   0.19   0.21   0.36   0.31   0.23   0.23   0.23
16   0.20   0.46   0.27   0.39   0.36   0.31   0.23   0.23   0.14
17   0.31   0.54   0.20   0.13   0.13   0.33   0.15   0.15   0.15
18   0.31   0.50   0.20   0.13   0.13   0.33   0.15   0.15   0.15
19   0.14   0.13   0.21   0.14   0.13   0.15   0.17   0.17   0.27
20   0.13   0.20   0.29   0.21   0.20   0.14   0.25   0.25   0.15
21   0.13   0.29   0.39   0.21   0.20   0.14   0.15   0.15   0.25
22   0.13   0.19   0.36   0.29   0.27   0.21   0.14   0.14   0.14
23   0.13   0.20   0.29   0.21   0.20   0.14   0.25   0.25   0.15
24   0.23   0.21   0.13   0.14   0.13   0.25   0.17   0.17   0.27
25   0.23   0.21   0.13   0.14   0.13   0.25   0.17   0.17   0.27
26   0.15   0.14   0.14   0.15   0.14   0.17   0.17   0.13   0.30
27   0.23   0.21   0.13   0.14   0.13   0.25   0.17   0.17   0.27
28   0.23   0.21   0.13   0.14   0.13   0.25   0.17   0.17   0.27
29   0.14   0.13   0.21   0.23   0.21   0.15   0.17   0.17   0.40
30   0.15   0.14   0.23   0.15   0.14   0.17   0.18   0.13   0.13

      10     11     12     13     14     15     16     17      18

1
2
3
4
5
6
7
8
9
10
11   0.29
12   0.20   0.43
13   0.42   0.46   0.29
14   0.24   0L53   0.20   0.39
15   0.45   0.50   0.39   0.55   0.50
16   0.45   0.39   0.29   0.31   0.31   0.46
17   0.25   0.21   0.21   0.33   0.23   0.15   0.36
18   0.25   0.21   0.21   0.33   0.23   0.15   0.36   1.00
19   0.17   0.33   0.23   0.15   0.50   0.27   0.27   0.30   0.30
20   0.15   0.21   0.21   0.14   0.23   0.15   0.25   0.17   0.17
21   0.15   0.42   0.31   0.14   0.33   0.25   0.50   0.27   0.27
22   0.14   0.39   0.13   0.13   0.31   0.23   0.33   0.15   0.05
23   0.15   0.21   0.21   0.14   0.23   0.15   0.25   0.17   0.17
24   0.40   0.23   0.14   0.36   0.15   0.27   0.27   0.30   0.30
25   0.40   0.23   0.14   0.36   0.15   0.27   0.27   0.30   0.30
26   0.30   0.15   0.25   0.27   0.17   0.30   0.13   0.20   0.20
27   0.40   0.23   0.14   0.36   0.15   0.27   0.27   0.30   0.30
28   0.40   0.23   0.14   0.35   0.15   0.27   0.27   0.30   0.30
29   0.27   0.14   0.14   0.25   0.15   0.27   0.17   0.13   0.13
30   0.30   0.15   0.15   0.17   0.17   0.18   0.13   0.20   0.20

      19     20     21     22     23     24     25     26     27

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20   0.30
21   0.44   0.40
22   0.40   0.50   0.50
23   0.30   1.00   0.40   0.50
24   0.20   0.18   0.18   0.17   0.18
25   0.20   0.18   0.18   0.17   0.18   1.00
26   0.22   0.33   0.20   0.18   0.33   0.57   0.57
27   0.20   0.13   0.18   0.17   0.18   1.00   1.00   0.57
28   0.20   0.18   0.18   0.17   0.18   1.00   1.00   0.57   1.00
29   0.20   0.18   0.13   0.27   0.18   0.50   0.50   0.57   0.50
30   0.22   0.20   0.20   0.13   0.20   0.38   0.38   0.43   0.38

      23     29    30

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29   0.50
30   0.33   0.38
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Title Annotation:Original Article
Author:Rawashdeh, Ibrahim Mohammad; Ghzawi, Abudel Latif; Rawashdeh, Nasab Q.; Khairallh, Kamal; Al-Tawaha,
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7JORD
Date:Jan 1, 2009
Words:3893
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