Analysis of Nepenthes Hybrids in Bukik Taratak West Sumatra by RAPD Technique

The Study about Analysis of Nepenthes Hybrids in Taratak Hill, Pesisir Selatan Sumatera Barat by RAPD Technique had been conducted from May until November 2011. The study was done to prove natural hybrids of Nepenthes by RAPD technique and to determine the relationship of natural hybrids of Nepenthes with both parental. The research method is survey and observation. Isolation of DNA carried by CTAB method with modifications and amplification carried out by PCR. Hybrids and parental relationship was obtained from Jaccard similarity coefficient and displayed in a Dendogram (family tree) and PCO analysis to look genetic variation was processed with the program PAST 2.10. Proof of the natural hybrid N. ampullaria × N. mirabilis can be done by the RAPD technique using the primers OPA 15, OPK 16, OPK 19 and OPP 15. Natural hybrids between N. ampullaria × N. gracilis using the primers OPA 15, OPK 19 and OPP 15. The eighth of natural hybrids N. ampullaria × N. mirabilis were closer to N. mirabilis, while both natural hybrids N. ampullaria × N. gracilis closer to the N. Ampullaria.


Introduction
Nepenthes is a unique plant [1][2][3][4][5][6]. The uniqueness and distinctiveness of Nepenthes is found in the character of the bag which is a modification of the end of the leaf, so that this plant is called the Pitcher Plant. Nepenthes have varying in shapes and colors. In Indonesia, in general this plant is better known as the Kantung Semar, but in some areas Nepenthes is also called by a local name, including kettles, monkey pouches and kuran-kuran. In West Sumatra, Nepenthes is known as Hill found that there were three hybrids in the Taratak Hill, namely hybrid N. ampullaria × N. mirabilis; N. ampullaria × N. gracilis and N. gracilis × N. mirabilis [8]. Hybrid testing with morphological character, at this time is not enough, because identification based on morphological characters has a very wide variation and can still be influenced by environmental factors and can not be done in juvenile or tillering [10] so it will be better to confirmed again with molecular testing.
RAPD technique is the simplest and fastest technique because it only requires amplification of the target DNA with PCR, without having to know the target DNA sequence. RAPD markers can be used in identification of varieties or strains, phylogenetic studies and genetic mapping [11]. RAPD is a technique that can be used to confirm natural hybrids [12].

Experimental Section 2.1.Tools and Materials
Tools needed in a sample collection are GPS (Global Positioning System) navigation tools, cutting tools and plastic collections. The material used for sample collection is young leaves (shoots) of plants and silica gel as sample preservatives. Equipment needed for the isolation of DNA, namely scissors, Mortar and Pestle, eppendorf 2 mL and 1.5 mL of sterile, spatula, waterbath, 10-200 mL pipette, 100-1000 mL pipette, vortex, Centrifuge max. 14,000 rpm, chemical hood , eppendorf tube stand (test tube rack), 37ºC incubator, evaporator and refrigerator.

2.2.Methods used 2.2.1.Sample Collectionin the Field
The sampling position was recorded using the GPS (Global Positioning System) navigation tool. A sample of putative hybrid leaves Nepenthes ampullaria x N. mirabilis, N. ampullaria x N. gracilis, N. ampullaria, N. mirabilis and N. gracilis, and male and female individuals from their respective parents (N. ampullaria, N. mirabilis and N. gracilis), were taken and stored in a collection of plastic filled with silica gel. Samples in silica gel are stored in a dry place, if silica gel has been pink, replaced with new silica gel, until the sample is used.

2.2.2.Isolation and amplification of DNA
DNA isolated from young leaves of putative hybrid and both parents. DNA isolation was carried out by modification of CTAB method, DNA isolation protocol developed by Doyle and Doyle [13]. DNA amplification was carried out according to Williams, et al. [14]. This study used 4 RAPD primers obtained from the BB Biogen Molecular Biology Laboratory, Bogor, namely OPA-15, OPK-16, OPK-19 and OPP-15. Primer refers to research onmolecular analysis Nepenthes with RAPD and RAPD Hybrid Analysis that has been done previously [15,16,17]. DNA electrophoresis was carried out based on methods found by Smithies with several modifications [18].

2.2.3.Natural Hybrid Analysis
Data were analyzed descriptively where hybrid assertion was based on the specific bands of both parents and their inheritance in natural hybrids. Analysis of natural hybrid kinship with both parents was carried out using cluster analysis and PCO (Principle Coordinate), where DNA bands were scored 1 for the presence of bands, and 0 for the absence of bands. Cluster analysis is done by calculating the value of genetic similarity based on the Jaccard similarity coefficient with the output in the form of dendogram carried out by themethod Paired Group. In addition to the cluster analysis,analysis was conducted Principle Coordinate (PCO)to determine the pattern of hybrid genetic variations with both parents. Cluster analysis and PCO were processed using the Analysis program using the Paleontological Statistics (PAST) program 2.10.

Results and Discussion
Natural hybrids Nepenthes found in Taratak Hill Pesisir Selatan are natural hybrids between species N. ampullaria and N. mirabilis; N. ampullaria and N. gracilis. Previous studies which reported that the natural hybrid of N. ampullaria x N. mirabilis was determined by looking at the character of the edge of the leaf which partly had coarse hairs that were rare and long and some had short, dense feathers, which were the characters of the mixture from the edge of the leaf N. ampullaria which is short-haired and tight with the edges of the leaves N.mirabilis which are rough and long hair which is rare and long. The lower sac is in the form of a urceolate like N. ampullaria but in the upper part of the tube, which pushes up to reach the lip of the sac. The character of the sac lips has a shape between the two parents, such as the wide and flat N. mirabilis lip shape of plus the shape of the sac N. ampullaria which curves long into the inside of the bag [19].
The natural hybrid form of N. ampullaria x N. gracilis is a form between the two parents, according to previous studies which found that the natural hybrid N. ampullaria x N. gracilis was determined by looking at the character of the pocket lips wider than the lips of N. gracilis, but narrower from the lips of N. ampullaria. The oval to almost round hybrid pocket cover, which is a mixed character of lanceate N. ampullaria the linear-sac lid and closes the sac N. gracilis orbicularis-ovate. The shape of a hybrid bag like N. gracilis with a few spotting [8].

| E K S A K T A : B e r k a l a I l m i a h B i d a n g M I P A ( S c i e n c e P e r i o d s E K S A K T A o f M I P A )
The identification of hybrids confirmed by looking at specific bands in both types of parents which inherited by individual derivatives [20]. Ribbons may appear only in hybrids, but are not in both of parents, and bands may appear only in one parent, do not appear in hybrids and other parent [10]. This was found in this study, with Primer OPA 15, the 1100 bp band is a band that only appears on N. ampullaria. with OPK 16 there is also a band, which is only on N. ampullaria male, which is at 500 bp and 850 bp. In OPK 19, there is only 1 band which is only owned by N. ampullaria male, which is at 1000 bp, and the band is only owned by N. mirabilis , which is at 700 bp. Crossing-over that occurs during meiosis may cause the loss of important loci, so that markers that appear on the elders do not appear in the offspring. Also on OPK 16 primer, a band that only appears on hybrids, which is at 100 bp, is a unique band. This unique band appears in the offspring which may be a result of recombination, random mutation or segregation that occurs on meiotic chromosomes during hybrid formation [15].
The Proximity among N. ampullaria x N. Mirabilis with parental. PCO analysis proves that some hybrids have proximity to the first type of parent and some hybrids have proximity to second type parents and some hybrids have a balanced proximity to both parents (figure 2).
Three primers used gave better results for identified hybrid N. ampullaria x N. mirabilis than hibrid N. ampullaria x N. gracilis. This can be assumed due to errors in the introduction of hybrid N. ampullaria x N. gracilis visually with morphological characters. In this study the number of primers used has provided good results in analyzing hybrids, but it would be better if we use more primers, so it might be possible to produce more varied and stronger ribbon and polymorphic patterns in analyzing hybrids.hybrid research Nepenthes might get better and more accurate results when using more RAPD primers.

The Proximity among N. ampullaria x N. Mirabilis with parental
PCO analysis proves that some hybrids have proximity to the first type of parent and some hybrids have proximity to the second type of parent and some hybrids have a balanced proximity to both parents. From axis 1 it can be seen that hybrids 1 and 3 with the RAPD technique have a closer relationship with the elders of N. ampullaria. From axis 2 it can be seen that hybrid 1 is closer to ampullaria maleand hybrid 3 is closer to N. ampullaria female. While we can assume that hybrid 1 has parental N.ampullaria maleand N. gracilis female and hybrid 3 have parental N.ampullaria female and N.gracilis male. But of course this assumption requires further research.

Conclusion
Based on the research conducted, it was concluded that the Nepenthes species found in Bukit Taratak Pesisir Selatan with the RAPD method proved to be true hybrids that occur naturally between N. ampullaria × N. mirabilis and N. ampullaria × N. gracilis. The natural hybrid relationship of N. ampullaria × N. mirabilis is closer to N. mirabilis, while the natural hybrid N. ampullaria × N. gracilis is closer to N. ampullaria.