Genetic Diversity of Ficus carica L. Based on Non-Coding Regions of Chloroplast DNA Anurug Poeaim1*, Supattar Poeaim1, Kasem Soytong2 and Tassnart Krajangvuthi3 Department of Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand 2 Major of Plant Pest Management Technology, Faculty of Agriculture, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand 3 Phratamnak Suan Pathum, Tumbon Bang Khayaeng, Amphoe Mueang, Pathumthani, 12000, Thailand 1 Abstract Ficus carica were analyzed for DNA sequence diversity in the non-coding region of chloroplast DNA. The trnL (UAA) intron sequences was used as genetic markers and establishing refined genetic relationships for differentiating F. carica collected from Phratamnak Suan Pathum originating from diverse geographical areas. Using the c and d primers, a single DNA band of approximately 550 bp was amplified from each fig cultivars. Twenty-one F. carica sequences from this study and 7 public sequences from the GenBank database were revealed the presence of 2 main groups. The first group is monophyletic branch composed by Ventura cultivar. All the remaining cultivars are ranged in the second cluster that comprises two sub-groups. Most of them were revealed a very low genetic diversity. The result was suggest that direct sequencing of the trnL (UAA) intron regions do not evolve rapidly enough to resolve relationships at these lower taxonomic levels. Keywords: Genetic Diversity, TrnL (UAA) Intron, Ficus carica 1. Introduction Ficus carica L., a deciduous tree belonging to the Moraceae family, is commonly known as fig tree. The fig is a typical Mediterranean fruit species which cultivated for its sweet fruits that are an excellent source of minerals, vitamins and dietary fiber. This fruits are consumed either fresh or dried and for industrial production such as jam and beverage. Different parts of the fig tree (leaves, fruits, bark, root and latex) are also used traditionally for their medicinal properties. F. carica is widely distributed in all the climatic stages and great diversity. More than sixty cultivars are listed in various reports [1-2]. Traditionally, the distribution of genetic diversity has been characterized using morphological markers such as leaf, fruit weight, shape and colors. However, these characters are either highly influenced by environmental conditions or limited to the fruit production season. Example, the leaf morphological characterization is difficult since the leaves of fig have difference shape [3]. *Corresponding author . Tel: 662-329-8000 ext 6223 Email: kpanurug@kmitl.ac.th The 8th International Symposium on Biocontrol and Biotechnology 163 In Thailand, there is one for fig cultivation for development of new typical agricultural products and their increased offer at local markets, introduction of figs into food processing industry, acquisition of new source of income for local growers and family farms. Recently, collection from traditional plantations has been initiated and conserved at Phratamnak Suan Pathum in Plant Genetic Conservation Project under the Royal initiative of Her Royal Highness Princess Maha Chakri Sirindhorn. Collected fig has great diverse of morphological characterization. Consequently, problems of synonymy and homonymy have been occurring. At the time of the above research, genetic diversity at the molecular level had not been investigated. The resulting information will contribute to the pool of background genetic information which may then facilitate the selection of a suitable conservation or breeding program. So, the main objective of this study was to determine the amount of genetic diversity and genetic relationships between varieties obtained from various parts of world as well as establish a molecular database for fig breeding programs. 2. Materials and Methods 2.1 Plant material Two sets of F. carica were used in this study. The first includes 21 cultivars that the fresh leaves were collected from Phratamnak Suan Pathum and the second, 7 public sequences of F. carica from the GenBank database. 2.2 DNA extraction and amplifications Genomic DNA was extracted from freshly harvested leaves using the Qiagen DNeasy plant extraction kit (QIAGEN) following the manufacture’s instructions. The trnL (UAA) intron was amplified using primers c (CGAAATCGGTAGACGCTACG)and d (GGGGATAGAGGGACTT GAAC) of Taberlet et al. [4]. Amplification reactions were carried out in 25 µl final volume of reaction mixture containing 200 µMol dNTPs, 0.8 pMol each primer, 10X PCR buffer and 1U Taq DNA polymerase (Biolabs, England) and 100 ng of genomic DNA. The thermocycler was programmed for an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min 30 sec, 2 min of annealing at 55°C; extension was carried out at 72°C for 3 min and final extension step at 72°C for 5 min. PCR fragments were separated using 1% agarose gels electrophoresis in 1XTBE buffer, stained with ethidium bromide and photographed on a UV transilluminator using a digital camera. The PCR products were purified and then used directly for sequencing. 2.3 Phylogenetic analysis All sequences were compared to others in GenBank using BLASTN and the best match recorded and selected. The entire DNA sequences of 21 F. carica isolates from this study and 7 public sequences of F. carica from the GenBank database were edited within Bioedit version 7.0.5.2. Nucleotide sequences were aligned using the ClustalX 1.83 software. The consensus trees were constructed the Phylip package version 3.6 with Neighbor-joining method by 1000 bootstrap resembling. Phylogenetic inference was performed and exposed using TreeView program. 3. Results and Discussion Molecular data are helpful in reconstructing phylogenetic relationships. The most widely used markers in plant are from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA The 8th International Symposium on Biocontrol and Biotechnology 164 (nrDNA). In recent, chloroplast DNA (cpDNA) is the most powerful tool for plant molecular systematics and phylogenetic relationships studies using universal primers following [4] both intron trnL (UAA) and intergenic spacer between the 3-exon of trnL (UAA) and trnF (GAA). Those chloroplast genes are now used routine because direct sequencing of polymerase chain reaction (PCR) products makes it relatively easy to obtain sequence data. So, the trnL (UAA) intron sequence was used as genetic markers and establishing refined genetic relationships. Twenty-one figs were amplified using the c and d primers; the designed PCR has permitted to generate banding profiles using templates of total cellular DNA. A unique fragment of approximately 550 bp was amplified from each fig cultivars. Sequencing directly from the purified PCR products was used to sequence analysis. Blast search was performed in order to confirm the identity of the sequences. Phylogenetic tree was constructed using neighbor-joining (NJ) analysis of the chloroplast sequences data. Relationships among cultivars of fig-tree, the dendrogram illustrated in Figure 1 has clustered the 28 cultivars into two main groups. The first group is monophyletic branch composed by Ventura cultivar. All the remaining cultivars are ranged in the second cluster that comprises two sub-groups. Sub-group 1 formed by the cultivars Celeste, Adriano, Marylane seedless, Fioroni umbrella, Bifara, Brown turkey, Paradiso nero and Fico gentile as well as Sawoudi (EU191023). The strong association at the DNA level between the Paradiso nero and Fico gentile cultivars. This study suggests that these cultivars are use to breeding program. Sub-group 2 contained about 18 cultivars that revealed relatively close relationship was also detected. So, this study uncovered some intraspecific diversity in Figure 1. Baraket et al. [5] was investigated the nucleotide variation of the trnL and trnF genes intergenic spacer non-coding region indicates that the plastid DNA of fig has been undergoing rapid expansion in their evolutionary history. On the other hand, genetic diversity based on trnL intron sequences has highly conserved which the pairwise sequence divergence ranged from 0.000 to 0.035 with an average of 0.012. The low level variation exhibited by fig cultivars is probably related to its slow evolution rate [6]. Molecular diversity of F. carica have been reported using various approaches, i.e. isozymes [7], simple sequence repeats (SSRs) [8-9], randomly amplified polymorphic DNAs (RAPD) [1, 10-13] restriction fragment length polymorphism (RFLP) [14] and amplified fragment length polymorphism (AFLP) [15] as well as sequence of ribosomal DNA [16-17]. These molecular markers can be effectively used to detect genetic diversity and the phylogenetic relationship between Ficus varieties. Cabrita et al. [15] was analyzed 11 Sarilop which is the main and standard cultivar for commercial dried fig production in Turkey by three molecular marker techniques: isozymes, RAPDs and AFLPs. Isozyme systems permitted the discrimination between Sarilop and Sarizeybek that used as a control. The use of 31 10-mer primers in RAPD analysis allowed splitting the 11 Sarilop into two groups of genetic similarity, but not to distinguish between all the clones. However, eight combinations of EcoRI/MseI primers in AFLP analysis were enough to clearly distinguish between all the Sarilop clones. The present study shows that cytoplasm DNA markers can be used to develop a molecular database for use in breeding program of the fig in the future. An understanding of genetic diversity of F. carica can provide insight into the management of this species. So, the enlarge number of markers by the use of other molecular methods are necessary in order to have a deeper insight into the identification key in this crop. On the other hand, morphological would be analyzes because of cultivars are selected by farmers mainly on the basis of agronomic traits such as fruits size and taste. The 8th International Symposium on Biocontrol and Biotechnology 165 Ventura Celeste Adriano Marylane seedless Fioroni umbrella Bifara Brown turkey 6 EU191023 Sawoudi 60 Paradiso nero 659 Fico gentile 1000 EU191014 Grichy Isfahan Italiano Pingo de mel 2 Qila saif Duaphine Sugar EU191017 Bither Abiadh 4 Black genoa 11 EU191005 Zidi1 141 EU191020 Dchiche Assal Fracazzano Conadria Horai 3 Fico umbrella 10 Milanzana 54 EU191024 Khalt 948 EU191007 Widlani Figure 1. Phylogenetic analysis on trnL (UAA) intron sequence of chloroplast DNA, 21 varities of F. carica comparative with 7 other varities from GenBank using bootstrap 1,000 replicates and Neighbour-joining algorithm. Bootstrap values are given at each branch point. 4. Conclusions The trnL (UAA) intron sequences was used as genetic markers and establishing refined genetic relationships for twenty-one F. carica and 7 public sequences from the GenBank database were revealed the presence of 2 main groups. 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