Sago palm is a starch-producing plant which is included in the family of Palmae and the genus of Metroxylon. This plant can grow in an environment where other plants cannot grow and resulting crops. The sago palm can tolerate salinity up to 171 mMNaCl for their growth because sago palm possessed mechanisms to limit the distribution of Na from the roots to the leaves [ 1 ]. Sago palm growth falters in the media with Al content reaches 200 ppm [ 2 ]. Potential of starch production of sago palm commodities is higher than other starch-producing plants. Starch production of sago palm was reported 3 to 4 times higher than the production of rice, corn or wheat and 17 times higher than the production of cassava [ 3 ]. [ 4 ] documented that the average productivity of sago palm each year up to 25 tons/ha. It makes sago become an important commodity that needs to be developed to meet the need for food and energy. Sago Starch has long been used by most of the local people in Papua as a staple food in the form of Papeda, Sinole, Kapurung, Sago Lempeng and Bagea [ 5 ]. Currently, a lot of variations of food made from sago starch, such as noodles, pastries, bread, cake, and brownies [ 6 ]
Sago palmsthat grow naturally and semi cultivated are naturally pollinated to form fruits and seeds, if not cut down to take the starch contained in the trunk. Sago palm pollination may occur as self-pollinated and cross-pollinated. Cross pollination leads to the expansion of genetic diversity due to recombination or segregation [ 7 ]. [ 8 ] reported that the type of sago palm pollination is cross-pollination, so the seeds that it is produced will diverse of their character. The phenotype characters will be expressed after the germination process to form seedling.
Information of plant genetic diversities is very important to be used for germ plasm collection and conservation. When germ plasm conservation activity is done, information of genetic diversities needed, especially from the natural habitat to carry out germ plasm conservation efficiently. A popular
2. Materials and Methods
2.1. Sample Collection
Young leaf of sago palm samples were collected from sago palm seedlings that is grown from seeds. The seeds were collected from one sago palm tree at the fruiting stages. A total 10 samples of 100 seedlings were selected and used in this study (Figure 1). Sago palm leafs were cleaned by using wet tissue with alcohol 70%. Approximately 0.5 gram fresh young leaves were used for isolation and extraction DNA. Isolation and extraction of total DNA from fresh sago palm leaf samples were conducted by using procedures as described by Geneaid DNA extraction kit (Geneaid, 2016). The total
2.2. PCR Amplification
PCR reactions were performed by using a machine Gene Cycler, Biorad. RAPD primers were used as follows OPA1 (5’-CAGGCCCTTC-3’), OPAW05 (5’-CTGCTTCGAG-3’), OPD08 (5’-GTGTGCCCCA-3’),
Figure 1. Performance of plant material used as sample in the study. Sprouting seeds (A) seedlings that is growing from the seeds after 10 months in the nursery were used as leaf sample for DNA isolation (B).
P01 (5’-GCGGCTGGAG-3’), OPA04 (5’-AATCGGGCTG-3’). The decamer random primers were provi- ded original sequences by Operon Technologies, Alameda, USA and synthesized by Integrated DNA Tech- nologies. Reagents used are Go-Taq-Green Master Mix kit, Promega. The PCR reaction with a total volume of 10 ml consisting of 3 ml ddH2O, 5 ml Go-Taq-Green mastermix, RAPD Primer 1 ml, and 1 ml of genomic DNA samples. Temperature profile used was 80˚C for 10 second, 94˚C for 2 minutes, 35 cycles consisting of three steps (94˚C for 30 seconds for denaturation, 40˚C for 60 seconds for annealing and 72˚C for 90 seconds for extension), the last extension at 72˚C for 5 minutes, and closed with a temperature of 24˚C for 1 minute.
2.3. Electrophoresis of PCR Product
Visualization of PCR products made with 2% agarose gel electrophoresis with a voltage of 80 volts for 1 hour. Each well contains 4 ml gel PCR product and 1 ml of loading dye. Staining of DNA molecules made by soaking the gel in a solution containing Ethidium Bromide (EtBr) for 20 minutes and visualized using UV-transluminator. Documentation DNA band performed using a digital camera.
2.4. Data Analyses
Polymorphism data analysis and construction of the phylogenetic tree is done using software NTSYSpc 2:02 (Applied Biostatistic). DNA bands of each RAPD marker transformed in the form of binary numbers 1 and 0. The number 1 indicates the present of DNA bands and 0 indicating no DNA bands. Dissimilarity matrix was calculated by using distance coefficient. The dissimilarity matrix was employed to construct phylogenetic by the Unweighted Pair-Group Method Arithmetic Average (UPGMA), using the Sequential Agglomerative Hierarchical Nested Cluster Analysis (SAHN-clustering, [ 12 ] and TREE program from NTSYS-pc, version 2.02 packages [ 13 ]. Bootstrap analysis with permutation 10,000 times were performed by using software Tools for Genetic Analysis (TFPGA 1.3).
3. Results and Discussions
3.1. RAPD Polymorphism
Polymorphism of RAPD amplification fragments by using five RAPD primers and performed in the PCR tools were resulted 26 fragments, 19 numbers of polymorphic fragment, 7 numbers of monomorphic fragment, and 8 numbers of genotype. Sample DNA fragments resulted by PCR were shown in Figure 2. High number of RAPD polymorphisms and genotypes were found in this observation. These results were similarly with polymorphism of sago palm in the previous study by using RAPD markers for revealing genetic relationship of natural stand of sago palm in Indonesia [ 14 , 15 ] by using RAPD markers for revealing relationship between geographical distribution and genetic distance of sago palm in Malay Archipelago. Figure 2 showed that the performance samples of DNA bands were amplified by using 5 primer sets. Numbers of fragment DNA band were amplified from each primer, and it was ranging from 3 to 9 fragments. Primers OPAW05, OPA01, and OPA04 were observed overall amplification DNA fragments it shown polymorphic whereas, primer P01 was resulted 3 DNA fragments polymorphic and 5 DNA fragments monomorphic and primer OPD08 was resulted 7 DNA fragment polymorphic and 2 DNA fragments monomorphic. Amplification of DNA sago palm by using those primers set was consistently resulting high polymorphism [ 16 - 18 ]. The polymorphisms of DNA fragments product by using RAPD markers were related to the variation of morphological phenotypic. Morphological characteristics of sago palm in the natural vegetation were observed in the previous study that were reported a large variation [ 19 ].
3.2. Genetic Variations
The Genetic distances of among individual samples were shown in the Table 1. Individual samples number S03, S04, and S07 were genetically the same because calculation the genetic distances in the Table
Figure 2. Performance of RAPD fragments by using 5 RAPD primers on 2% agarose gels. Primer OPA04 (A) and primer OPD08 (B). Individual sample indicated by S01 to S10.
Table 1. Calculation value of distances among sample based on Nei’s unbiased (1978) distance.
Notes: S01 to S10 indicated individual samples.
Figure 3. Dendrogram construction of samples based on 5 RAPD markers by using UP-GMA clusters. S01 to S10 indicated individual samples.
1 were shown zero and the biggest distances between individuals were sample S01 and S10 with the values 0.8602. Sample S01 and S10 were genetically large differences. The previous study were reported that the sago palm in the natural habitat show a lot of variation based on morphological characteristic [ 19 ], growth and yield production [ 20 ] Chloroplast DNA [ 17 ], and specific expression gene [ 18 ].
Analysis of phylogenetic trees based on the pattern of polymorphism of RAPD markers was done by using UPGMA method. The dendrogram construction of 10sago palm samples is presented in Figure 3. Genetic relationships of sago palm progenies that have been grown were divided into four groups in the level dissimilarities 40% at the position of coefficients 0.40. Group 1, 3, and 4 just one in individual and Group 2 associated with seven individuals. The molecular observations were indicated that the progenies of sago palm derived from natural pollination were high diversities. The genetic distances among individual progenies were presented in the Table 1. These features indicated that sago palm in the natural condition was occurred cross-pollination process. In the previous studies were reported that the pollination type of sago palm is crosspollinated, therefore the characters of sago palm progenies were caused diverse [ 8 ]. These characters will be expressed after the seeds germination process. Generally, pollination of sago palm occurred a cross pollination because male and female flower mature in different of time period [ 21 ].
The progenies of sago palms that derived from naturally pollinated were genetically varied based on RAPD markers. Variations occurred in the progenies of sago palm indicated that the sago palm were estimated naturally cross-pollinated, the nresulting fruits and seeds with genetically differences.
Grateful go to the management of MP3EI project with SPPK No.162/SP211/LT/ DRPM/III/2016 from DRPM Directorate General of Strengthen Research and Development (DGSRD) Indonesia for providing financial support in the Fiscal Year 2016. Thanks also to all who were contributed and reviewed in this work.