Analysis genetic diversity of rombeng pines using RAPD markers

The tourism industry in Indonesia continues to grow from year to year, as evidenced by the expansion of tourism villages. In the context of tourism, the term’resources’ refers to everything that has the potential to be developed to directly or indirectly support tourism. The natural potential of the Rombeng pine forest (Pinus sp.) in Bantaeng Regency, South Sulawesi, has become a tourist attraction for the surrounding community and visitors from outside the city. This pine species has a trunk characteristic that distinguishes it from scattered pine in general. This trait is manifested by grayish skin and a reddish hue that can be exfoliated. Based on this, an examination of the genetic diversity of pine species in the Bantaeng district was conducted. Utilizing Random Amplified Polymorphic DNA (RAPD) markers and Darwin 6. 0 as a research methodology. This study indicates that the diversity value (HE) of Pinus species is 0.46, which is relatively high. The near category of based genetic distance is dominated by genetic kinship, and the closest genetic distance is 0.04 while the farthest is 0.80.


Introduction
Tourism in Indonesia is one of the industries with the greatest potential to support the country's economy and its diverse tourist destinations.Hal has been the driving force behind the development of tourism in Indonesia to this point.In the context of tourism, resources are defined as anything that has the potential to be utilized directly or indirectly to support tourism.South Sulawesi's Bantaeng Regency has natural potential in the form of the Rombeng Pine Forest, which is being developed as a tourist destination.
The only naturally occurring endemic pine species in Indonesia is Pinus merkusii, which grows in Aceh, Tapanuli, and Kerinci on the island of Sumatra [1].Pine (pinus merkusii) is a gymnosperm or open-seeded plant with needle-shaped leaves and a spruce or cone-shaped tree canopy.Pine is a fast-growing species that can thrive in marginal (less fertile) soils and does not require a specific growing environment.Almost every part of the tree can be utilized, including the trunk, from which sap can be extracted.Construction, furniture, proofreading rods, pulp, and long fiber paper can all benefit from wood products.Its coolie material can be used as fuel, while the ash is added to fertilizer blends [2].
The primary distinction between pine rombeng and P. merkusii is that the bark on pine rombeng's trunk is grayish and can be peeled off, whereas P. merkusii's trunk is brownish, slightly cracked, and difficult to exfoliate on the skin.The level of genetic diversity is one of the determinants of breeding and conservation strategies' success.A population with high genetic diversity, resistance to disease and extreme climate change, and the capacity to live in sustainable conditions [3].It has been demonstrated that molecular markers can provide superior accuracy and reliability in plant genetic diversity analysis.The creation of various types of molecular markers, such as isoenzymes, restriction fragment length polymorphisms (RFLP), simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD).RAPD markers are one of the DNA markers that are highly efficient, simple, and inexpensive for identifying individual differences [4][5][6].
On this basis, it is anticipated that the research conducted will yield knowledge about genetic diversity useful for genetic breeding and conservation development and programs.This study's objective is to examine the genetic diversity of Pinus species in Rombeng Pine Forest, Bantaeng Province.

Location and timing of research
This study will be conducted between November 2020 and November 2021.This study was conducted in genetic diversity analysis utilizing RAPD markers.In Bonto Lojong Village, Ulu Ere District, Bantaeng Province, samples were taken.At the Biotechnology and Tree Breeding Laboratory, the Silviculture Laboratory of the Faculty of Forestry, and the Laboratory of Chemistry and Soil Fertility, Faculty of Agriculture, Hasanuddin University, Makassar, genetic diversity were observed and analyzed.The number of samples used was 30 trees.This research examined the first few stages of DNA isolation, primary selection, and genetic diversity analysis.The DNA isolation process is carried out by the Cetyl Trimethyl Ammonium Bromide (CTAB) method with modifications [7].

Primary selection
The primary selection is conducted to identify RAPD primers that generate tape markers based on the clarity of the bands generated and the number of loci obtained.Primary selection by performing multiple PCR reactions with multiple primers under identical conditions and using the same DNA sample in order to determine the optimal conditions and the degree of band variation produced by each primer.The primary selection is conducted to select polymorphic, amplifiable primers and determine the optimal annealing temperature.Table 1.Amplification names for RAPD primary selection.

Genetic diversity analysis
The amplified primer will be used for the entire sample's PCR reaction.Amplification of DNA using a Sensoquest PCR instrument.The PCR stage begins with an initial denaturation process at 95°C for 5 minutes, followed by 35 cycles of initial denaturation at 94°C for 60 seconds, specific primary pasting (temperature adjusted to each primary pair) for 60 seconds, elongation stage at 72°C for 60 seconds, and final propagation at 75°C for 10 minutes.For long-term use, PCR results are stored at 4 or -21 ° C. Electrophoresis was used to separate the amplification results using 2% agarose and TAE1x buffer.Electrophoresis was performed at 100V for sixty minutes.Using a UV transilluminator and a digital camera, the electropherogram is observed and recorded.The obtained result appears as a band in order for the Ribbon to display the allele present at a particular locus.Each primer used contains a unique locus.The ribbons that appear are numbered in accordance with their dimensions.The size of the largest band is 1, and if there is no ribbon, it is 0. The presence or absence of the tape is determined by manually observing the electropherogram photograph.The data is then tabulated and processed with the software Darwin 6.5 to determine its kinship and genetic variation.This formula is used to calculate the heterozygosity value [8]: Heterozygous : qi =( Information: Pi = 1-qi He = 1 -pi 2 Description: ܰ .‫݂‬ ‫ݏݐ݁ܪ‬ represents the number of heterozygous individuals in a population: N Represents the sum of all individuals in a population. The value of polymorphic information content (PIC) is calculated using the following formula [9]: PIC = 2 be (1-be) Information: PIC = polymorphic information content Fi = allele frequency

Genetic diversity analysis
Utilizing RAPD markers, genetic analysis was used to examine genetic variation and kinship relationships among pine sp.varieties.The genetic diversity parameters consist of the number of detected alleles, the average value of expectation heterozygosity N a, and the average value of observational heterozygosity He (calculated using the Microsoft Ho) Excel application.The parameter data was derived from the results of manual scoring on all samples that had undergone polyacrylamide gel electrophoresis and the PIC online program in order to generate the information data in table 2. Table 2 shows the 7 primers used in the analysis of genetic diversity.The primer was chosen by considering the clearest, brightest and polymorphic band visualization results.The highest Na content in the genetic diversity parameter was OPQ 20 and OPG 06 with 7 while the lowest was OPAC 12 with 3. The He value that had the highest value in the genetic diversity parameter was OPK 20 with a value of 0.46 while the lowest was OPAC 12 is as much as 0.20.Based on research [10], it shows that the genetic diversity of trees plus seeds is at a moderate level (He=0.508).
For analyzing the genetic diversity of pines, the average PIC value of 0.44 where this results in hal 7 primers is instructive.A high PIC value indicates that the primer can detect a significant number of alleles [11].The informative value of a primer is determined according to [12], which states that a primary is classified as informative if its PIC is less than 0.3.On this basis, the entire sample was extremely informative regarding the primary effectiveness of this study.By analyzing the band patterns depicted based on the amplification results entered into Darwin's software, it is possible to determine the kinship relationships between individuals in the entire sample using dendrograms and genetic distances 6.5.The analysis of 30 samples of Pinus sp.indicates the presence of two major genetic clusters within the pine family.The first cluster contains 29 individuals, whereas the second cluster contains only one.The same cluster is characterized by nearly the same genotype name and is generally formed from the same population, so its kinship level is closer, whereas a genotype with a very different name but a high degree of kinship is likely to have inherited its genetic material from the same parent but has been dispersed to different locations, hence its different names [13].The dendrogram of kinship relationships of pine individuals (Pinus spp.) reveals that individuals 9 and 6 share a high degree of genetic similarity, whereas the genetic distance between individuals is based on Figure 3.This indicates that individuals 18 and 19 have the shortest genetic distance at 0.02, while the genetic distance between the most distant individuals is 0.80, found between individuals 3 and 29.A high genetic distance value denotes low genetic similarity between individuals, and vice versa.A PIC value close to 0.5 is an exceptionally effective value for distinguishing individuals.The value of genetic diversity is high when He is less than 0.5, and it is low when He is equal to 0.5.[9].Using RAPD markers, the genetic study of pine stands identified in the Hasanuddin University Education Forest aims to determine the genetic differences between pine individuals in stands.The analysis of pine DNA using 10 RAPD markers revealed the greatest genetic distance to be 0.9630 and the smallest to be 0.2698.At 0.489, the genetic diversity of pines in the Unhas Education Forest is comparatively high, some like in research Senna spectabilis [12], Phalaenopsis [15], Red Wood [16], Nilgirianthus ciliatus [17].According to [18], genetic distance correlates with the kinship between individuals as indicated by dendrograms.Close kinship denotes a small genetic distance, whereas distant kinship denotes a large genetic distance.

Analysis of the overall kinship relationship of individuals
On the basis of genetic distances, it is demonstrated that the presence of variation between individuals in a population can be attributed to the genetic mixing of one parent tree surrounding it as a result of hybridization.Inbreeding can also contribute to a decline in population size.The mating system influences the magnitude and pattern of genetic variation in forests, according to [19].Relational or closed mating systems decrease the proportion of heterozygotes and increase the number of weak offspring or those with high homozygosity.

Conclusion
Analysis of the genetic diversity of safety in Pinus sp trees revealed two clusters.This suggests that close genetic kinship dominates the category of genetic kinship.

Figure 2
displays the results of the analysis of the kinship relationship of individual pine rombeng (Pinus sp.) using the Darwin application in this study conducted in Bantaeng Regency.

Table 2 .
Parameters of genetic diversity Caption: Na= Number of Alleles He = Heterozygosity of Expectations, PIC = Polymorphism Information Content