Phylogenetic Tree of Kuantan Cattle by DNA Barcoding

Hidayati, Misrianti R, Ali A. 2016. Phylogenetic tree of Kuantan cattle by DNA barcoding. JITV 21(1): 41-48. DOI: http://dx.doi.org/10.14334/jitv.v21i1.1351 Kuantan cattle is one of local beef cattle breed of Riau Province which its origin was unknown. Kuantan cattle are commonly found in Indragiri Hulu and Kuantan Singingi Regency. Based on phenotype characterizations, kuantan cattles are similar with pesisir cattle (West Sumatera beef cattle). Historically, kuantan cattle were pesisir cattle brought by “minang” immigrants (Immigrant from West Sumatera) to this region. The purpose of this study was to analyze the origin of the kuantan cattle through genetic diversity analysis using DNA barcode. DNA barcode used was Cytochrome oxidase subunit I gene which was found in the mtDNA. DNA isolation was done on 25 kuantan’s blood samples and 18 pesisir blood samples. Amplification of COI gene segment used Polymerase Chain Reaction technique. The forward primer sequence used in this study was F’5 TTCTCAACCAACCATAAAGATATTGG-3’ and the reverse primer sequence used was reverse 5’TAGACTTCGGGGTGTCCAAAGAATCA-3. It squeezed kuantan and pesisir sequence 5711 6420 base (GeneBank accession number NC_005971) with length by 710 bp. Analysis result of sequence using MEGA 5.2 Program showed that there were 6 polymorphic sites establishing 7 haplotypes on kuantan cattle and 9 polymorphic sites establishing 12 haplotypes on pesisir cattle. Based on genetic distance and phylogeney tree, kuantan and pesisir cattle were in same group with Bos indicus. Mutation in the COI gene segment in this study was too small and was not able to distinguish the difference of those breeds. The result of neighbor joining analyze indicated that kuantan cattle origin was from Bos indicus just like pesisir cattle.

. This kuantan cattle has been reared with extensive and semi intensive system for a long term.
According to Misrianti (2014), dominant color of kuantan cattle was white-brown with upward-curved horns and white legs.Phenotypically, kuantan cattle are similar with pesisir cattle and historically, kuantan cattle were pesisir cattle brought by "minang" immigrants (from West Sumatera) to this region.The development of every species under its particular natural ecosystem, environmental, and socio-economic conditions has led to each having its own specific genetic characteristics (Yang et al. 2013).Domestication of livestock species and a long history of migrations, selection and adaptation have created an enormous variety of breeds (Hailu & Getu 2015).The most domestic cattle in Indonesia belong to Bos taurus or Bos indicus while Indonesian Bali cattle was domesticated form banteng (Bos javanicus) (Kusdiantoro et al. 2009) One of conventional methods applied in animal genetic resources assessment is morphological markers.Morphological markers, normally refer to external animal characteristics (animal's phenotype), which can be obtained by direct visual observation and measurement.Animal's phenotype is determined by its genetic, environment and interaction of both.Evolution of farm animal genetic resources through morphological markers based on subjective judgments and descriptions and the conclusions reached were often not completely accurate.Information on genetic diversity and origin of kuantan cattle has not been reported.
Determination of breed or line of livestock by Indonesian government was aimed to guarantee the utilization and conservation of animal genetic resources in a sustainable manner, as well as legal protection providing for breed or lines of the existing livestock.Management of animal genetic resources is essential for the achievement of global food security emphasizing global food production, improvement of productivity and food availability.People may use genetic resources to develop animal production to meet food requirement.However, sufficient genetic markers for evaluating population structure and other aspect of animal genetic resources require genetic diversity assessment.One method for population characterization was DNA barcoding (Yang et al. 2013;Hailu & Getu 2015).A DNA barcode was a short DNA sequence from a standardized region of the mitochondrial DNA gene, mitochondrial cytochrome c oxidase I (COI), used for species identification (Hebert et al. 2003).
COI gene was a gene that responsible for final step in phosphorylation before the establishment of adenosine triphosphate (ATP) (Sutrisno et al. 2013).COI gene had a conserve sequence of mitochondrial DNA on livestock (Mueller 2006) and might be used to analyze the origin of livestock (Wilson 2010) and was suitable for evolutionary studies (Lunt et al. 1996).The advantages of COI gene as a DNA barcode were (1) a relatively short length of the gene which was about 648 bp, (2) relatively stable and not prone to mutation, (3) variability was very low 1-2%, (4) much amount of copies was easily to be amplified than genes -gene nuclear DNA (Sutrisno et al. 2013).Some research reports using DNA barcode on birds (Herbert et al. 2004), Indonesian local cattle (Febriana 2011), chicken (Gao et al. 2011), cetartiodactyla (Zein & Fitri 2012), bali cattle (Syed-Shabthar et al. 2013) and Indonesian local buffalo (Saputra et al. 2013) has been reported.
This study was aimed to obtain phylogenetic tree of kuantan cattle and to study whether pesisir cattle was the origin of the kuantan cattle based on DNA barcoding.

Materials
Blood samples of more than two years old kuantan cow (n=25) from Kuantan Singingi (Riau Province) (Figure 1) and more than two years old pesisir cow (n=18) from central of superior cattle-forage breeding of Padang Mengatas, West Sumatera (Figure 2) were collected from jugular vein using 3 mL syringe.Those blood samples were preserved in EDTA and kept in room temperature from October to November 2015 for laboratory analysis.

DNA isolation was done in Animal Molecular
Genetic Laboratory, Bogor Agricultural University on November 2015.Genomic DNA was extracted using Phenol-chloroform Technique (Sambrook et al. 1989) and modified by buffer lysis cell use (250 µL 1 X STE, 40 µL SDS and 10 µL Proteinase-K).The DNA was purified by adding 40 μL 5M NaCl, 400 μL phenol chloroform and 400 μL CIAA (Chloroform Iso Amyl Alcohol) and precipitated using 40 μL 5M NaCl and 800 μL ethanol absolute.The precipitation was washed once by adding 800 μL of 70% ethanol and centrifuged at 12.000 rpm of speed for 5 minutes.Ethanol was discarded and evaporated.The precipitated DNA was dissolved in 100 μL of 80% TE (Elution buffer).2013).Position of forward and reverse primer in PCR product of COI gene was shown in Figure 3.Samples were initially denaturized at 93°C for 3 minutes and followed by 35-36 cycles of denaturation at 93°C for 30 second, annealing at 60°C for 30 second and extension at 72°C for 30 second.Final extension was at 72°C for 5 minutes.Master Cycler Personal 22331 Eppendorf was used for PCR amplification.PCR products were then separated on 1.5% agarose/1 x TAE, stained with 2.5 μL of ethidhium bromide (EtBr) and calibrated with 100 bp ladder marker.Electrophoresis chamber was run on 100 volt power supply for 35 minutes.Finally, the gel was visualized under Gel Doc.Forty three PCR product samples were then subjected to direct sequence (forward and reverse) analysis by dideoxy sequencing in ABI 3730 XL automated DNA sequencer at the First base laboratory Malaysia via commercial service on January 2016.

Quality of DNA isolated and PCR amplification of COI gene
Qualitative method for identification, separation and purification of DNA fragments was by agarose gel electrophoresis (Fachtiyah, et al. 2011).DNA which might migrate on agarose gel or polyacrylamide gel was placed in a buffer solution which electrified in chamber.Negatively DNA will be moving positive direction.The success of DNA isolation characterized by a single band, bright and cleared in each well (Figure 4).M X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 13 14   1).DNA concentration of pesisir cattle was 29.9597.45ng/µL with purity level of 1.71 to 1.80.Good purity levels of DNA isolated were 1.8 to 2.0.If the value exceeds of 2.0, the solution tested was still contaminated by membrane protein or other compounds so that the levels of plasmid DNA obtained was not pure.If it was less than 1.8, it means that too much ddH2O was taken while DNA taken was too slightly.The success of PCR process was determined by accurately and level of concentration of DNA used as a template.The range of concentrations of DNA used in the PCR process was 25-50 ng/mL.
Primer has amplified COI gene of kuantan cattle and pesisir cattle successfully.It was indicated by the appearance of a single band at 710 bp (Figure 5).Annealing temperature was 60 0 C for 45 seconds.

Genetic distance and phylogeny tree of COI gene on Kuantan and Pesisir cattle
Results of the analysis of genetic distance showed that kuantan and pesisir cattle might be grouped into Bos indicus because it had a genetic distance of 0.000 in all haplotypes, except on K Haplotype on pesisir cattle (0.002).Genetic distance of kuantan cattle and Bos taurus was in the range of 0.10 to 0.12.This result explains that based on the DNA barcode, kuantan cattle is in the same cluster with Bos indicus as well as pesisir cattle, aceh cattle and PO cattle.Cattle breeds have been developed in different ways depending on regional climates, nutritional conditions and selection for different purposes.Genetic drift also contributed in the process of breed differentiation.In very closely related breeds, the number of mutations cannot explain the observed genetic variation even when highly mutable DNA sequences are used.
The result was in accordance with Mitchell et al. (2010) who said that the genetic diversity of COI gene in the Bovidae was between 0.0 -1.92% (average 0.245) and 9.77% in average between the species.Genetic distance in the Bos, Capra, and Ovis genus was 3.5%, 0.0%, 0.003% respectively.Genetic distance between javanicus -indicus and javanicus -taurus species was 0.066% and 0.014%, respectively (Zein and Fitri, 2012).In this study, genetic distance between kuantan and Bos indicus cattle was 0.00 and between kuantan and Bos Taurus was among 0.10-0.12.Genetic distance in height of COI gene between the species was low.It showed that COI gene was effective for identification at the level of species, genus and family and was appropriate to be used as a DNA barcode.For small size (N=50) population and mutation rate of β = 10 -3 , the difference between computed inbreeding value assumed that mutation was small (less than 7 % of true value) and mutations might be neglected during 200 generations (Laval et al. 2002).
Reconstruction of phylogeny tree of kuantan cattle used neighboor-joining method.The result showed that all samples of cattle kuantan were in the same cluster with the Bos indicus with gene bank access number of KF 952 285, KF 952 284, KF 771 228 and Bos Taurus with gene bank access numbers of JX 18056.1.It was vary from Bos Taurus access number of HQ 860420.1,HM 102290.1,KF 771228.1 and FJ 958 333 with the genetic distance of 0.001 (Figure 6).This research showed that based on the maternal line, the ancestor of kuantan cattle was Bos indicus as well as pesisir cattle.

Figure 4 .
Figure 4. Qualitative test of DNA isolated

Figure 6 .
Figure 6.Phylogeny tree of Kuantan cattle by Neighboor-Joining Method .27 cm; and shoulder height 99.19 cm (Ministry of Agriculture Republic of Indonesia 2014).Kuantan cattle were found along the Kuantan River (from Kuantan Singingi Regency to Indragiri Hulu Regency).Total population of kuantan cattle in Indragiri Hulu Regency (5950 heads) was more than in Kuantan Singingi which were 2386 heads (Department of Animal Husbandry and Animal Health of Riau Province kuantan bull had body length by 103.78 cm; chest girth by 126.22 cm; and shoulder height by 99.28 cm, while kuantan cow had body length by 102.35 cm; chest girth 123

Table 1 .
Concentration and Purity Level of Isolated DNA DNA Figure 5. PCR amplification product of COI Gene (710 bp); M = DNA ladder 100 bp, X2, X3..Xn = samples codes Concentration of isolated kuantan's DNA was 27.45-121.45ng/µL with a purity level of 1.64 to 1.81 (Table

Table 2 .
Type of haplotype of Kuantan and Pesisir cattle