Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 82 http://www.agriculmag.uodiyala.edu.iq/ PHYLOGENETIC RELATIONSHIPS AMONG (GRAPEVINE) Vitis TAXA BASED ON RANDOM AMPLIFICATION OF POLYMORPHIC DNA (RAPD-PCR) MARKERS Aseel K. Al- Anbari 1 Mohanad W. Al_ Zubaidy Najm A. Al- Zubaidy Ammar A. Sultan Shereen M. Mahmood Department of Biology, Education College for Pure Sciences, Univ. of Diyala, Iraq 1 Correspondin auther: aseelalanbari@yahoo.com ABSTRACT Phylogentic relationships among seven genotypes of Vitis were analyzed by RAPD-PCR markers, DNA of fresh young leaves was extracted from each sample for RAPD analysis. Performed with 20 decamer primers selected six primers that showed clear results. A total sixty eight bands were produced out of which forty eight bands were polymorphic, The dendrogram reveals that grapevine taxa separated to main groups (A and B), the first group includes the most of taxa were Vitis hissarica Vass., V. hissarica subsp. rechingeri, V. sylvestris Fl. Bed., V. vinifera L. (Native), and V. vinifera var. sativa Beck., while group B contains 2 species were V. coignetiae Planch., and V. berlandieri Pulliatt. The taxa V. hissarica and V. hissarica subsp. rechingeri have been recorded closest genetic distance matrix among taxa under study with similarity range 0.11. The results of this study were offered an important data in taxonomy and may be can be a phylogenetic key to isolating the taxa. Moreover it is provide basic information for future genetic studies such as gene expression profiling. Key words: Grapevine, hylogenetic, RAPD, genetic distance. INTRODUCTION Grapevine (Vitis vinifera L.) belong to Vitaceae family, one of the anciently and momentous perennial crops in the global (Bodea et al, 2009; Nagaty et al, 2011). Townsend and Guest (1980) reported three main species in Iraq, and confirmed there were no wild species found in Iraq, because the genus extensively cultivated. Vitis occur in the tropical and subtropical regions (Soejima and Wen, 2006). Classical procedures of identification, based on botanical, morphological, and ampelographic characters consider instability due to the influence of internal and external environment (Bodea et al, 2009). In addition, morphological method requires extensive field features and evaluation (Nagaty et al, 2011). Molecular Received: 19/6/2016 Accepted: 9/3/2017 http://www.agriculmag.uodiyala.edu.iq/ mailto:aseelalanbari@yahoo.com Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 83 http://www.agriculmag.uodiyala.edu.iq/ markers have more advantages compering with morphological and biochemical markers (Kamali et al, 2010). The molecular systematic studies give us deeper light of genetic structures. Fingerprinting markers have been an impotence role for many purposes in molecular biology, such as analyzing of granitic diversity for classification of the cultivars and germplasm collections, and clearing the phylogenetic relationships among taxa, or closely related species. The identify sequence of nucleotides in the DNA of plants significantly increased our understanding of plant’s families evolution, (Carlson and Holsinger, 2010). Vitaceae has been considered a confusing and complicated taxonomic group with unclear genetic lines (Lombardi, 2000). The phylogeny of Vitis has been widely employed in the last decades, among them random amplified polymorphic DNA RAPD (Ye et al, 1998 ; Tamhankar et al, 2001 ; Bodea et al, 2009 ; Nagaty et al, 2011), amplified fragment length polymorphism AFLP (Cervera et al, 2000 ; Ergul et al, 2006), restriction fragment length polymorphism RFLP (Bourquin et al, 1993), inter simple sequence repeat ISSR (Sabir et al, 2009), simple sequence repeat SSR (Bowers et al, 1996 ; Sefc et al, 1999), and chloroplast of DNA cp (Trias-Blasi, 2012) also have been used to analyze the genetic diversity to identification in grapevine cultivars. RAPD technique has been widely used in DNA gene mapping isolation of phylogenetic relationships of many ranks, because low cost, tiny plants material, and its ability to reveal high degree of polymorphism it has successfully been applied as a molecular technique for cultivar identification within many plant families (Karataş and Ağaoğlu, 2010 ; Nagaty et al, 2011; Alanbari et al, 2014 ; Alanbari et al., 2015). However rare data about the genetic diversity within grape species in Iraq. This study was attempted to investigate and identify of genetic diversity and relationships among grapevine cultivars, as well as systematic phylogenetic analysis of these taxa based on DNA profiling using RAPD technique can using as phylogenetic taxonomic key to isolate taxa . MATERIALS AND METHODS Plants materials: Fresh young healthy grape leaves of seven taxa of genus Vitis were V. vinifera L. (Native), V. sylvestris Fl. Bed., V. hissarica Vass., V. coignetiae Planch., V. berlandieri Pulliatt., including 1 subspecies V. hissarica subsp. rechingeri, and 1 variety V. vinifera var. sativa Beck collected during May 2015 from Diyala province. To identify the taxa we used the taxonomic keys that http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 84 http://www.agriculmag.uodiyala.edu.iq/ published in flora, such as flora of Iran (Parsa, 1951), flora of Turkey (Davis, 1975), and flora of Iraq (Townsend and Guest, 1980). DNA extraction: Approximately 50 to 100 mg of young fresh leaf tissue put in 1.5 ml tube, homogenized the tissue using liquid nitrogen with a conical hand tissue grinder. Genomic DNA was isolated from leaf samples using the procedure described by DNeasy Plant Mini Kit Protocol (Bioneer, Korea). Screening of PCR: A total of twenty different 10mers RAPD primers were tested in this study (Table 1) supplied by Bioneer company were screened, six primers which had been shown indicated results of band patterns, multi master mix were used, the thermos profile for the PCR reaction was: 95 °C for 5 minutes, then 35 cycles of 95 °C for 30 seconds, 37 °C for 1 minute, and 72 °C for 5 minutes. Genotypes were visualized on 1% agarose gel, 1x TBE, stained with 0.5 mg ml-1 ethidium bromide, with 1500 bp ladder (SibEnzyme Ltd. Russia) and 100 v for 45 min visualized and photographed under a UV transilluminator. Data analysis: The amplified bands were scored as 1 or 0 based on presence or absence of a band, within the size range between 110-1150 base pairs (bp). RAPD matrix analyzed by NTSYS-pc statistical package version 2.1. The data matrix was used to calculate the genetic similarity within taxa based on Jaccard’s similarity coefficients, and a dendrogram displaying relationships among the 7 genotypes was constructed by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA(. Table 1. The sequences of twenty RAPD primers used in study including those produced amplified Primer name Sequence (5´- 3´) Primer name Sequence (5´- 3´) OPA-01 CAGGCCCTTC OPC-13 AAGCCTCGTC OPA-02 TGCCGAGCTG OPF-08 GGGATATCGG OPA-03 AGTCAGCCAC OPF-14 TGCTGCAGGT OPA-13 CAGCACCCAC OPG-01 CTACGGAGGA OPA-18 AGGTGACCGT OPG-02 GGCACTGAGG OPB-01 GTTTCGCTCC OPM-04 GGCGGTTGTC OPB-02 TGATCCCTGG OPM-15 GACCTACCAC OPB-03 CATCCCCCTG OPO-07 CAGCACTGAC OPC-06 GAACGGACTC OPZ-01 GAGCCCTCCA OPC-12 TGTCATCCCC OPZ-03 CAGCACCGCA http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 85 http://www.agriculmag.uodiyala.edu.iq/ RESULTS AND DISCUSSION 68 amplified RAPD bands outcome from this study ranging from 110 bp to 1.15 kb in size were shown from the 7 grape genotypes. Due large numbers of polymorphism, we can predict that a strong relationship among genotype under study. The number of RAPD bands varied from 7 (primer OPM-04) to 17 (primer OPO-07). Forty eight polymorphic bands were outcome (Table 2), (Figure 1). Table 2. The Total number and size range of amplified bands obtained for each primer AN= amplification number; PM= Polymorphic bands; MM= Monomorphic bands, %PM= PM/ANx100, % MM= MM/AN x100 According to Trias-Blasi et al (2012) the phylogentic studies of Vitis were unresolved. The numerous bands based on each primer depends on sequence of primer and extent of variation in specific genotype (Chan and Sun, 1997 ; Shukla et al, 2006 ; Shiran, 2007). The dendrogram reveals of genetic linkages among the seven Vitis genotypes (Figure 2) shown that grapes taxa were mainly separated into two basic groups with a similarity value at 0.44, the first (group A) consisted of five Vitis taxa, it had divided to three clusters, V. sylvestris separated from other four taxa with similarity value 0.2, while V. vinifera and V. vinifera var. sativa which separated by value similarity at 0.15, but V. hissarica and V. hissarica subsp. rechingeri have been recorded closest genetic distance matrix among taxa under study with similarity range 0.11, (group B) contained two taxa were distinct genotype were V. coignetiae, and V. berlandieri that separated at the similarity value at 0.32. These two taxa isolated from other genotype. The general similarity ratio were found between 0.11-0.44. This results were associated with Sabir (2009) but disagreed with other researchers were they reported the similarity ratio were 0.3-0.9 in Turkish grape cultivars (Ergul et al, 2002 ; Karataş, 2005 ; Ağaoğlu, et al, 2006 ; Primer name Sequence (5´- 3´) AN Size range of bands(bp) PM % MM % OPA-18 AGGTGACCGT 12 110- 910 8 66.7 4 33.3 OPF-08 GGGATATCGG 10 200- 700 7 70 3 30 OPF-14 TGCTGCAGGT 10 150- 1090 5 50 5 50 OPM-04 GGCGGTTGTC 7 250- 650 4 57.1 3 42.9 OPM-15 GACCTACCAC 12 195- 800 11 91.7 1 8.3 OPO-07 CAGCACTGAC 17 155-1150 13 76.5 4 23.5 Total 68 48 20 http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 86 http://www.agriculmag.uodiyala.edu.iq/ Karataş and Ağaoğlu, 2010) may be this ascribable to strong relationship among the taxa under study. Different authors agreed that genetic diversity was attributed by two factors, the geographic origin of varieties, morphological characters and the somatic mutations (Ulanovsky, 2002 ; Ergul et al, 2002 ; Sabir, 2009 ; Karataş and Ağaoğlu, 2010). Our DNA finger printing study was shown no environment’s power or biotic conditions effecting on genetic diversity among grapevine genotype, this is match with obtained by Nagaty (2011), and his results confirmed the two cultivars showed high similarity because they had the same fruit characters, but speciation pressures may be caused 4% differences, this result agreed with our results that observed the strongest homogeneity between V. hissarica and V. hissarica subsp. rechingeri Although the seven taxa were collected from the same farm but they have different genetic profile during the time (Nagaty, 2011) as V. coignetiae, and V. berlandieri isolated from other genotype. So it could be considered as a reservoir of alleles useful for breeding, because the divergent genotypes may had a reliable breeding value ( Gwanama et al, 2000), or had substitution averages and high levels of gene rearrangements. Fig. 1. RAPD profile obtained with 6 primers. M-Marker1500-100; 1- V. sylvestris; 2- V. vinifera var. sativa; 3- V. vinifera; 4- V. coignetiae; 5- V. berlandieri; 6- V. hissarica; 7- V. hissarica subsp. rechingeri http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 87 http://www.agriculmag.uodiyala.edu.iq/ Fig. 2. Dendrogram of the7 Vitis taxa tested However, from this study we can recognize that the new data were combined with existing botanical and molecular data it will be significant potential for genetic mapping, and identification in grapevine taxa. Moreover it considering a phylogenetic key can be useful tool in systematic studies to isolate taxa as well as an effective tool to understand evaluating gene flow in order to identify the taxa that could be further evaluated. These results indicate to interesting and high degree of correlation among grapevine taxa. REFERENCES Al-Anbari, A. K, N. Kanawapee, T. A. Al-Kazragi, H. Al-Jewari, A. Al- Mashhadani, S. Barusrux, P. Pornpongrungrueng and P. Theerakulpisut. 2014. Genetic diversity of Citrus (Rutaceae) in Iraq based on random amplified polymorphic DNA (RAPD) markers. African journal of agricaltral research. 9(11): 1012- 1019. doi: 10.5897/AJAR2013.8306. Al-Anbari, A. K., M. W. Al-Zubadiy and W. M. Dawood. 2015. Genetic diversity of some taxa of Cucurbitaceae family based on “RAPD” markers. Advances in Life Science and Technology, 37: 7-11. ISSN 2224-7181 (Paper) ISSN 2225- 062X (Online) www.iiste.org. Ağaoğlu, YS, H. Karataş and D. Degirmenci. 2006. Molecular characterization of some local (İskilip-Çorum) Anatolian grape cultivars (Vitis vinifera L.). 9th International Conference on Grapevine Genetics and Breeding. Udine, Italia. Acta Horticul., 827: 207-210. Bodea, M., D. Pamfil, R. Pop, and I. F. Pop. 2009. Use of random amplified polymorphic DNA (RAPD) to study genetic diversity among Romanian local http://www.agriculmag.uodiyala.edu.iq/ http://www.iiste.org/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 88 http://www.agriculmag.uodiyala.edu.iq/ vine (Vitis vinifera L.) cultivars. Bulletin UASVM Horticulture. 66(1): 17-22. ISSN 1843-5254; Electronic ISSN 1843-5394. Bourquin J. C., L. Otten and B. Walter. 1993. Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera. Theoretical and Applied Genetics, 87: 157–162. Bowers J. E., G. S. Dangı, R. Vıgnamı and C. P. Meredıth. 1996. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome., 39: 628–633. Calson, J. E. and K. E. Holsiner. 2010. Natural selection on inflorescence color polymorphism in wild Protea populations: the role of pollinators, seed predators and inter trait correlations. Amr. J. of Botany. 97: 934- 944. Chan, K. and M. Sun. 1997. Genetic diversity and relationships detected by isozyme and RAPD analysis of crop and wild species of Amaranthus. Theor. Appl. Genet. 95: 865- 873. Cervera M. T., J. A. Cabezas, E. Sanchez-Eschrıbano, J. L. Cenıs and Martınez- Zapater. 2000. Characterisation of genetic variation within table grape varieties based on AFLP markers. Vitis. 39: 109–114. Doi 10.1600/ 036364412X656437. Davis, P. H. 1975. Flora of Turkey and the East Aegean Island. University of Edinburgh press. 1: 507- 508. Gwanama, C., MT. Labuschangne and AM. Botha. 2000. Analysis of genetic variation in Cucurbita moschata by random amplified polymorphic DNA (RAPD) markers. Euphatica. 113: 19-24. Ergul, A., K. Kazan, S. Aras, V. Cevık, H. Celık, and G. Soylemezogl. 2006. AFLP analysis of genetic variation within the two economically important grapevine (Vitis vinifera L.) varietal groups. Genome. 49: 467–475. Ergül, A., B. Marasalı and Y. S. Ağaoğlu. 2002. Molecular discrimination and identification of some Turkish grape cultivars (Vitis vinifera L.) by RAPD markers. Vitis. 41: 159-160. Kamali, M., A. Ahmadikhah, M. Pahlavani, M. Dehghan and F. Sheikh. 2010. Advances in Applied science research. 1(3): 180-186. Karataş, H. 2005. Molecular analysis of Diyarbakır Region’s Grapevine germplasm by RAPD (Random Amplified Polymorphic DNA) technique. Ph. D. thesis (unpublished). Ankara University, Ankara, Turkey. http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 89 http://www.agriculmag.uodiyala.edu.iq/ Karataş, H. and Y. S. Ağaoğl. 2010. RAPD analysis of selected local Turkish grape cultivars (Vitis vinifera). Genetics and Molecular Research. 9(4): 1980-1986. Doi: 10.4238/vol9-4gmr926. Lombardi, J. A. 2000. Vitaceae: geˆneros Ampelocissus, Ampelopsis e Cissus.Flora Neotropica: 80. New York: New York Botanical Garden. Nagaty, M. and S. El-Assal. 2011. Molecular characterization and genetic relationships among some grape (Vitis vinifera L.) cultivars as revealed by RAPD and SSR markers. European J. of Experimental Biology. 1(1): 71-82. Parsa, A. 1951. Flora De Iran. Imprimerie Danesh, Tehran. I(2): 1535- 1537. Radwan, S. A. 2014. Molecular disscrimintion and genetic relationships between some cultivars of Cucurbitapepo spp, pepo using random amplified polymorphic DNA (RAPD) analysis. Afr. J. of Biotechnology. 13(11): 1202- 1209. Doi: 10.5897/AJB2012.3007. Sabir, A., S. Tangolar, S. Buyukalaca and S. Kafkas. 2009. Ampelographic and molecular diversity among grapevine (Vitis spp.) Cultivars. Czech J. Genet. Plant Breed. 45(4): 160–168. Sefc KM., F. Regner, J. Glössl and H. Steinkellner. 1998. Inheritance of RAPD markers in an interspecific F1 hybrid of grape between Vitis quinquangularis and V. vinifera. Vitis., 37: 20. Shukla, S., A. Bhargava, A. Chatterjee, A. Srivastava and S. Singh. 2006. Genotypeic variability in vegetable Amaranth (Amaranthus tricolor L.) for foliage yield and its contributing traits over successive cuttings and years. Euphytica. 151: 103- 110. Soejima, A. and J. Wen. 2006. Phylogenetic analysis of the grape family (Vitaceae) based on three chloroplast markers. Am. J. of Botany. 93: 178–187. Tamhankar, S. A., S. G. Patil, and V. S. Rao. 2001. Assessment of the genetic diversity of some important grape genotypes in India using RAPD markers. Vitis. 40: 157–161. Townsend, C. and E. Guest. 1980. Flora of Iraq. Ministry of agriculture and agrarian reform, Baghdad, Iraq. 4(1): 443-449. Trias-Blasi, A., A. John, N. Parnell and R. Trevor. 2012. Multi-gene region phylogenetic analysis of the grape family (Vitaceae). Systematic Botany. 37(4): 941–950. http://www.agriculmag.uodiyala.edu.iq/ Diyala Journal of Agricultural Sciences, 9(special Issue): 82-90, 2017 Al-Anbari et al 90 http://www.agriculmag.uodiyala.edu.iq/ Ulanovsky, S., Y. Gogorcena, F. Toda and J. Ortiz. 2002. Use of molecular markers in detection of synonymies and homonymies in grapevines (Vitis vinifera L.). Sci. Horticul. 92: 241-254. Ye G. N., G. Soylemezoglu, N. Weeden and B. Reısch. 1998. Analysis of the relationship between grapevine cultivars, sports and clones via DNA fingerprinting. Vitis. 37: 33–38. على عالمات التضاعف العشوائي للدنا اعتمادا Vitis العنب العالقة الوراثية بين مراتب جنس شيرين محمد محمود عمار احمد سلطان نجم عبد هللا الزبيدي مهند وهيب الزبيدي 1سيل كاظم االنباريأ ، العراقجامعة ديالى ، كلية التربية للعلوم الصرفة،قسم علوم الحياة aseelalanbari@yahoo.comالمسؤول عن النشر: 1 المستخلص تقنية تفاعل البلمرة التسلسلي اشكال وراثية لجنس العنب تم تحليلها بواسطة 7العالقة الوراثية بين الدنا من اوراق يانعة طرية لكل عينة لتحليل التضاعفات العشوائية. تم العمل بواسطة واستخلص ،العشوائي حزمة من 48. كانت ج ملموسةاوضحت نتائوادئ ب 6 فقط منهابادئات ) ذات عشر قواعد ناتروجينية( 20 حزمة متباينة االشكال. اظهر الشكل ان مراتب العنب انفصلت الى مجموعتين اساسية )أ و ب( 68اصل :هيمراتب الاحتوت المجموعة االولى اغلب المراتب تحت الدراسة وكانت و Vitis hissarica Vass., V. hissarica subsp. rechingeri, V. sylvestris Fl. Bed., V. vinifera L.(Native), V. vinifera var. sativa Beck., ين من بترتبينما انفردت المجموعة الثانية ب . كانت اقرب معدل للعالقة الوراثية V. hissarica subsp. rechingeri و V. hissarica العنب وهما اعتبارها مفتاح تصنيفي لعزل المراتب النباتية مهمة للتصنيف ويمكن ه الدراسة بيانات ـذوفرت ه. 1.11 هي . كما انها وفرت معلومات اساسية للدراسات الوراثية المستقبلية للتعبير الجيني المسافة الوراثية. ،العشوائي التسلسلي البلمرة تفاعل تقنية ،التقارب الوراثي ،العنب الكلمات المفتاحية: http://www.agriculmag.uodiyala.edu.iq/ mailto:aseelalanbari@yahoo.com