DNA Extraction of Sumatran Striped Rabbit from Tissue Samples

The Sumatran Striped Rabbit (Nesolagus netscheri ) is likely a naturally rare, which is a protected animal based on Government Regulation Number 7 of 1999 and renewal of Permen LHK No. P.106 which are categorized as Data Deficient by the IUCN (International Union of Conservation of Nature) since 2019. Samples were obtained from Pagar Alam, South Sumatra. Most commonly found at elevations above 600 m in montane and sub-montane primary habitat. Molecular genetic characterization of sumatran striped rabbit is to know genetic information and genetic identification of N. netscheri. Primers 12S rRNA were used in this study to characterize Sumatran striped rabbit. A 1 band were detected ranged from 900 to 1000 bp. Molecular markers represent reliable tools which may have a great impact in rabbit breeding and genetic improvement of rabbits. Molecular markers on Nesolagus netscheri is expected to tools in the identification of Sumatran striped rabbits from South Sumatra.


Introduction
Sumatran striped rabbit (Nesolagus netscheri) is the endemic species of Sumatran [18] [6]. Nesolagus netscheri is found at an altitude between 600-1,600 m [3]. The observations have been reported since 1972, and most recently is on February 2017 in the area of Gunung Raya Wildlife Reserve [14]. Record from Gunung Leuser National Parks [3], Bukit Barisan Selatan National Parks [1] [7]. This species will be increasingly threatened if its natural habitat decreases due. This is interesting because information about N. netscheri very instrumental in conservation efforts [9].
Classification of the status of the Sumatran Striped Rabbit has been difficult. In 1994 the species was assessed as Endangered, in 1996 it was assessed as Critically Endangered, and in 2008 it was assessed as Vulnerable. These changes in status back and forth are reflective of the complete lack of information on the ecology of the species that has persisted over that time. Between 1996 when the species was assessed as Critically Endangered, and 2008 when the species was assessed as Vulnerable, there were only a handful of additional records of the species, all consisting of simple reports of occurrence [8].
Nesolagus netscheri is included in the list of protected animals based on Government Regulation No. 7 of 1999, Regulation of the Minister of Environment and Forestry Number P.106 of 2018 and includes animals in a Data Deficient status based on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species [8].
12S rRNA marker is a very good genetic marker for the study of kinship relationships between mammals [11]. Several studies have been carried out with genetic markers of 12S rRNA, such as in Lagomorpha [4] and in several other mammals [7] [16] [11], in addition in mammals, genetic markers of 12S rRNA have also been carried out on Insecta [12] [5]. Analysis of 12S rRNA gene sequences can be used for species determination and authentication of meat samples [13] and species identification [10] [19] The Sumatran rabbits and Annamite rabbits are morphologically similar, but genetic data indicate that they have been isolated for millions of years. Assuming a steady rate of divergence over time at this gene, the Sumatran and Annamite rabbits would have been diverging genetically for approximately 8 million years [15]. Molecular markers on Nesolagus netscheri is expected to help in the identification of Sumatran striped rabbits from South Sumatra.

Tissue Collection
The research sample is tissue (muscle) N. netscheri from South Sumatra, preserved with absolute ethanol. Samples were obtained from the local community, dead on

DNA Isolation
The stages of DNA isolation are based on the procedure for using the Wizard® Genomic DNA purification Kit from Promega. A total of 20 mg of N. netscheri tissue was put into a 1.5 mL tube and 275 µL of Digestion Solution Master Mix was added to each tube.

Purifikation
Move the supernatant from the 1.5 mL tube into the Wizard®SV Minicolumn that has a 2 mL Collection tube installed. Samples were sentrified at a speed of 13,000 rpm for 3 minutes then the centrifuge solution was discarded and the minicolumn was placed in a new 2 mL collection tube.
Then the DNA washesed by adding Column Wash Solution (CWA; with 95% ethanol added) as much as 650μl to the minicolumn and then centrifuged again at 13,000 rpm for 1 minute. The centrifuge solution is discarded and put back in the collection tube. Repeat this step with a total of 4 washes, and re-install the 2 mL collection tube, then centrifug for 2 minutes at 13,000 rpm to dry the matrix in the minicolumn.
Next the Wizard®SV Minicolumn was transferred into a new sterile 1.5 mL tube and added with 250 µL Nuclease-Free Water which was previously incubated at 600 C for 30 minutes. After that, the solution was incubated at room temperature for 2 minutes and centrifuged at a speed of 13,000 rpm for 2 minutes. The results obtained in the form of DNA in a 1.5 mL tube that has been isolated, store it in the freezer at a temperature of -20° C to -70° C.

DNA Amplification by PCR
Total DNA from isolation is used as printed DNA for the amplification process. Primers for amplifying 12S rRNA genes are 12SR and 12SL [17]. Primary attachment location for amplifying 12s rRNA genes (

Analysis of Amplification Results using Electrophoresis 2.5.1 Agarose Gels 2%
As much as 40 grams of agarose was weighed, then put into a 300 ml erlenmeyer. Next, 200 ml of TAE 1x solution was added and dissolved by heating it using a Hot plate. After the agarose solution is not too hot, 5 µL of gel staining is added, and homogenized, then the agarose solution is poured into the mold provided. The comb is installed to make a a well in the gel, then allowed to stand Saputra et al, 2019 | 3. Results and Discussion 48 for 15 minutes so that the gel solids. After the gel solids the comb is removed and a well is formed in the gel. The gel is completely immersed in a TAE 1x solution on the electrophorator and is ready for use for electrophoresis.

PCR Product Electrophoresis
DNA samples from PCR, loading dye and markers were prepared. The agarose that has been made is inserted into the electrophorator. Then with a micropipette, as much as 2 µL of DNA sample and 1 µL of loading dye are mixed first on the parafilm paper and put in the 2nd well until the last well. The 1st well was filled with 1 µL marker. The electrofarator is closed, then the voltage is set at an 80 Volt voltage for 45 minutes, migration direction from pole -to +, and the ON button is pressed on the machine. After DNA migration is complete, the machine is turned off. Gel electrophoresis results were then observed and photographed using gel documentation.

Results and Discussion
Usage of biotechnology in rabbit's significantly to the development and enforcement of genetic improvement programs [2]. The study suggests that PCR can be successfully utilized for detecting molecular genetic markers for rabbit's such as sumatran rabbit. These markers (fingerprints) providing an easy and rapid tools for characterization, identification and sustainable use in breeding programs. Molecular markers were used in the present study to obtain fingerprints for Sumatran striped rabbit. 12S rRNA primers were used in this study to characterize N. netscheri. Sample 1 DNA amount was too low to be detected, there is also multiple band observed, detected band ranging from 900 to 1000 bp, samples 2 and 3 did not have a band (Figure 2). The results of present studies can provide basic molecules information for future researchers. These results indicated efficiency of PCR techniques in the characterization of rabbit genotypes. Molecular markers on N. netscheri is expected to help in the identification.

Conclusion
The use of methods for the identification and characterization of genotypes is essential for rabbit protection. This study supplies comprehensive approaches for studying the genetically molecular characterization of Sumatran striped rabbits which can help the genetic development of rabbits. PCR techniques are effective methods for detecting DNA markers in Sumatran striped rabbits. These markers are useful for estimating genetic distances and relationships among other rabbits.