Genetic diversity analysis of Bitti (Vitex cofasssus), native species Indonesia based on Inter Simple Sequence Repeat (ISSR) marker

Bitti (Vitex cofassus) is a native species in Indonesia and is a superior wood in South Sulawesi. This type is a hardwood that is sought after and exported in large enough quantities. A narrow genetic base requires joint efforts to develop plant varieties, so it is necessary to carry out selection activities to obtain a superior diversity of traits. The most advanced method in assessing genetic diversity is a molecularly based approach using the Inter Simple Sequence Repeat (ISSR) marker. Germplasm exploration was carried out in Pattiroang Village, Kajang District, Bulukumba Regency, South Sulawesi Province. A total of 30 samples were extracted using the Genomic DNA Mini Kit (plant) method with modifications. Ten primers tested obtained six primers that produced bright polymorphic bands, namely UBC 820, UBC 822, UBC 823, UBC 824, UBC 827, and UBC 830. The highest He value is in the UBC 824 primary with a PIC of 0.48, while the lowest He values is in the UBC 827 primer with a PIC value of 0.15. The visualization of the dendrogram shows two large clusters, each consisting of a diverse number of individuals. It indicates a relatively moderate kinship between the types of V. cofassus.


Introduction
Bitti (Vitex cofassus) is a particular type of wood in South Sulawesi.Based on its botanical description, V. cofassus comes from the family Verbenaceae with a height of up to 40 m, pale bark, and hardwood.V. cofassus is a native species of Southeast Asia, covering the territories of Indonesia, Papua New Guinea, Malaysia, Thailand, and Vietnam [1].This type is a hardwood that is sought after and exported in large quantities.The wood is solid and durable, with a density of 700-800 kg/m -3 .Freshly cut wood has a rough aroma and is difficult to process with preservatives.
In South Sulawesi, more precisely in Pattiroang Village, Kajang District, Bulukumba Regency, the type V. Cofassus is widely used to houses, boats, and household appliances.The community widely plants V. cofassus on agroforestry land or secondary forest areas [2].The need for high use with good wood quality puts this type in great demand.V. cofassus in South Sulawesi is still grown with conventional propagation techniques.This is undoubtedly feared to reduce the rate due to inbreeding which will affect the decrease in crop productivity.Therefore, it is necessary to know and understand about the genetic potential of the bitti population to be used as material and development in the future.Knowledge of genetic diversity and the determination of the next step is the primary goal of any activity of repair and improvement of the peel of plants, for which the collection of information about genetic diversity becomes the first step that must be carried out.The most advanced method used in genetic diversity research is a molecularly-based approach [3,4].A molecularly-based approach is used to interpret the genome arrangement or identify DNA/RNA fragments of organisms based on sequence differences that indicate the presence of polymorphisms between different individuals within a species [5].
Some of the markers that are widely used in molecular research are the Inter Simple Sequence Repeat (ISSR) markers.This marker is based on Polymerase Chains Reaction (PCR), which has been tested to be quite efficient in analyzing plant genetic diversity and is used in assessing the variety of germplasm Fields [6].The existence of research on the genetic diversity of the V. cofassus type using ISSR markers is considered essential to be carried out as a first step in preparing a database as a reference in plant breeding programs, correction of varietal defects, and development of new varieties.

Plant material
Germplasm was explored in Pattiroang Village, Kajang District, Bulukumba Regency, South Sulawesi Province.Leaf samples were taken randomly in community plantation areas.Leaf samples were fresh leaves and not damaged to obtain good DNA purity during the extraction process.

DNA extraction
Genome DNA was extracted using the Genomic DNA Mini Kit (Geneaid plant) method with minor modifications.The mini kit (plant) is designed to be simpler for isolating genome DNA (gDNA) from various plant tissues.The Mini Kit contains a high concentration of salt (chaotropic salt) capable of removing water molecules in the solution.The mini kit is specially designed to adsorb DNA and empty carbohydrates, polyphenols, and other plant metabolites so that DNA purity can be obtained [7].The DNA extraction process was carried out at the Biotechnology and Tree Breeding Laboratory, Faculty of Forestry, Hasanuddin University, Makassar.
A total of 0.3 grams of leaves were finely ground, weighed 0.01 grams into the tube, then homogenized using 400 μl GP1 buffer and five μl RNase A, the function of adding this solution is to help degrade the cell wall in the sample.Tube containing the solution was incubated using a water bath at a temperature of 65°C for 60 minutes.The incubated sample was added 100 μl GP2 buffer and incubated using ice for 10 min.The tube was centrifuged at a 10,000 G (global) speed for 5 min, this is done to provide for the solution according to its specific gravity which is divided into filtrate and substrate.The filtrate is then transferred to a 2 ml column, and the tube filter is then centrifuged at the rate of 10,000 G for 1 minute.The supernatant was added to a 750 μl GP3 buffer, flipped, moved into the GD column, and centrifuged for 2 minutes.The supernatant is then discharged, 400 μl W1 buffer is added to the GD column, centrifuged for 1 minute, then the liquid is removed.GD columns are added 600 μl wash buffer and then centrifuged for 1 minute.Then the fluid is removed.The GD column is recentrifuged for 3 minutes this stage is carried out to obtain good quality and quantity of DNA.
The extraction DNA purification process is carried out by adding 100 μl of elution buffer that has been warmed and then incubated at room temperature for 5-10 minutes, then centrifuged at a speed of 10,000 G for 1 minute.The GD column is then discharged.The refining results are then stored in the freezer by adding three μl of RNase.This is the final stage of DNA extraction and isolation

Primer selection and amplification of ISSR markers
The quantity test of the extracted DNA was carried out using a Qubit 3.0 Fluorometer (Thermo fisher scientific).Qubit working was made with dsDNA reagent as much as 1 μl and qubit buffer as much as 199 μl then the results from qubit working were taken as much as 199 μl + 1 μl DNA sample.The results of the quantity test are then selected to determine the primary match that can produce a bright and clear 1230 (2023) 012076 IOP Publishing doi:10.1088/1755-1315/1230/1/0120763 DNA band.The type of primer used is ISSR (inter-simple sequence repeat), this primer was chosen because it has high reproducibility when compared to RAPD (Random Amplified Polymorphic DNA) primers.The various ISSR primers used are UBC-810, UBC-813, UBC-814, UBC-820, UBC-822, UBC-823, UBC-824, UBC-827, UBC-830, UBC-868.The selected primer is a polymorphic primer that makes a bright band.Genetic diversity testing was performed using PCR (Polymerase chain reaction), then visualized on the agarose gel.

Data analysis
The visualization results on the agarose gel are in the form of DNA bands representing alleles in the locus, and each primer used represents the locus.Scoring is done by assigning a value of 1 based on the length of the band that appears and 0 if there is no band.The data were tabulated and analyzed using Darwin 6.0 software to determine individuals' relationships and degree of genetic variation.Heterozygosity is calculated by: qi = Frequency of absent allele; pi = Frequency of dominant allele Polymorphic Information Content (PIC) was calculated by [8]:

Sampling location
The sampling of V. cofassus is in the area of mixed-community plantations.More details can be seen in Figure 1.

DNA extraction
The results of genome DNA extraction using the Genomic DNA Mini Kit (plant) method with minor modifications showed promising results for the V. cofassus type.The extraction results were tested using the Qubit 3.0 Fluorometer (Table 1).  1 above shows the purity level of the extracted DNA that varies from randomly tested V. cofassus samples.This level of DNA purity can be influenced by the secondary metabolite compounds of the extracted sample, such as polysaccharides, lipids, and polyphenols, or it can also be from extraction chemicals, duration of incubation, and employability skills [9].The ISSR marker was chosen because it has very polymorphic, flexible, and modifiable properties.It consists of 1-6 nucleotides in one primer universally used for phylogenetic analysis [10].A total of 10 primers were tested, and six primers were obtained that produced bright polymorphic bands.The six primers selected for genetic Diversity analysis were UBC 820, UBC 822, UBC 823, UBC 824, UBC 827, and UBC 830, with annealing temperatures of 51.0, 47.0, 48.1, 48.5, 53.0 and 52.7 respectively, these temperatures are determined according to the primary bond and template DNA (Table 2), at this temperature the primer was able to stick well, respectively.

Primer selection and amplification of ISSR markers
The use of molecular markers such as ISSR in the study of genetic diversity with the method of measuring genetic distance is based on the interpretation of loci/allele (allelic) models in various food crops or forestry crops [11,12].These markings require primary selection and application that will provide clear, different, reliable, and sufficient information to study the diversions between individuals/species.In this study, the number of polymorphic loci detected per primer varied by primary, Figure 2.  The results of electrophoresis visualization showed the diversity of DNA bands in each allele of V. cofassus.The DNA bands represent alleles in the locus, and each primer used will define the locus.The variations in the DNA bands that appear will represent the diversity or difference between each individual tested.
This study assessed six primers capable of determining the genetic diversity of the type of V. cofassus.All the primers used were to produce polymorphic and bright bands, with a DNA fragment size between 300-1,100 bp (Figure 2).A well-amplified ISSR primer will make high-quality polymorphic DNA bands informative for distinguishing genetic diversity [13].
The success of a marker in distinguishing polymorphic levels is expressed by polymorphic information content (PIC).Another parameter used to assess the success of a marker in determining individuals is the heterozygous value (He).These parameters are calculated manually from the results of the PCR band amplification total score and tabulated into the expected Heterozygosity formula [13,14].3 shows six primers that produce polymorphic bands with the highest He values in UBC 824 primers with PIC of 0.48, while primers with the lowest He values, namely in UBC 827 primers with PIC values of 0.15.PIC is a parameter that reflects the level of preliminary information.Values for PIC are classified into three, namely PIC ≤ 0.25 (low polymorphism or has a low informative level), 0.25 ≤ PIC 0.5 (medium polymorphism), and PIC ≥ 0.5 (high polymorphism or very informative primary) [13].The UBC 827 primary has a low PIC value meaning that this primer is not able to provide vital information; for the primer UBC 824, UBC 30, UBC 20, UBC 22, and UBC 23 categorized for the medium class, this value can provide information on the genetic diversity of V. cofasssus.

Genetic distance and dendrogram
Plant evolution is a form of adaptation to the environment influenced by diversity and genetic distance within the population.Genetic distance describes variations in a population caused by genetic mixing derived from crosses [15].The variety of plant genetic resources allows plant restorers to develop new and superior cultivars with better characteristics [16].
The   Understanding diversity and genetic relationships are essential for genetic conservation and tree breeding, some research in the Indonesian rice germplasm [17].This information is necessary for exsitu conservation to determine the number of populations and individuals the people that must be collected to maintain the established fundamentals of genetic diversity [18,19].The results of the dendrogram visualization and calculation of pic values above show a relatively high kinship relationship between types of V. cofassus.Each individual does not have the same DNA.The DNA sequence will always be different, even in elders and saplings.To some extent, these differences make up genetic diversity, or what is known as polymorphism.The degree of polymorphism of an organism is said to be formed by demographic fluctuations and can be in the form of changes in the biotic or abiotic environment field [20].

Cluster 1 Cluster 2
In this study, it emphasizes efforts to interpret the number and structure of genetic diversity of V. cofassus in the Pattiroang Village area, Kajang District, Bulukumba Regency, South Sulawesi Province, in the future; these results can be a reference in considering the possibilities that can arise as a result of climate change, biogeographic changes, habitat fragmentation, ecological disturbances, and human intervention [4,21].

Conclusions
A total of 10 ISSR primers were selected, and six primers were obtained that could produce clear and bright polymorphic bands, namely UBC 820, UBC 822, UBC 823, UBC 824, UBC 827, and UBC 830 primers.The results of dendrogram visualization and calculation of PIC values from the 30 samples tested above showed a relatively moderate kinship between types of V. cofassus.

Figure 1 .
Figure 1.Sample distribution map V. cofassus.yellow dots represent each individual who sampled the observation.
relationship between individuals is described by a dendrogram capable of showing a correlation with the genetic distance between individuals.Based on dendrograms and genetic distance calculations, individuals 8 and 9 have the lowest genetic distance values of 0.2, while the farthest genetic distance values are shown for individuals 10, 14, 16, 17, and 22, which is 0.50.Close relatives have a low genetic distance, while distant relatives have a high genetic space.

Figure 3
Figure 3 shows the kinship relationship of the type V. cofassus.Of the 30 individuals, V. cofassus is divided into two large clusters, each consisting of diverse individuals.The first cluster is divided into two sub-clusters, where individual 1 has a distant kinship with the individuals in the first cluster.The closest distance is indicated in individuals 8 and 9.This suggests a similarity between individuals that the same elders can cause inbred crosses (inbreeding) or to narrow a population size.The second cluster also showed 29 individuals who did not merge with the other groups.The closest distance in the second cluster is shown in individuals 21 and 30.Understanding diversity and genetic relationships are essential for genetic conservation and tree breeding, some research in the Indonesian rice germplasm[17].This information is necessary for exsitu conservation to determine the number of populations and individuals the people that must be collected to maintain the established fundamentals of genetic diversity[18,19].The results of the dendrogram visualization and calculation of pic values above show a relatively high kinship relationship between types of V. cofassus.Each individual does not have the same DNA.The DNA sequence will always be different, even in elders and saplings.To some extent, these differences make up genetic diversity, or what is known as polymorphism.The degree of polymorphism of an organism is said to be formed by demographic fluctuations and can be in the form of changes in the biotic or abiotic environment field[20].

Table 1 .
Results of DNA quantity analysis extraction from genomic DNA mini kit using qubit 3.0 fluorometer.

Table 2 .
ISSR primers and the nucleotide sequence.