Identification of a Differentiation Factor of Indonesian Ongole Cattle Breeds Based on Microsatellite Markers and Mitochondrial DNA

This study was conducted to identify the differentiation factor of Indonesian Ongole cattle breeds (Sumba Ongole and Ongole Grade) based on the 12 microsatellite markers and Cyt b gene polymorphism. A total of 50 blood samples (25 samples for each cattle breed) were used in this study. The multiplex DNA fragment analysis was conducted for allele identification based on the microsatellite markers. The haplotype identification (based on the mitochondrial DNA) was conducted using restriction fragment length polymorphism (RFLP) analysis with three restriction enzymes i.e. HinfI, HaeIII, and XbaI. Twelve microsatellite loci in this study revealed high polymorphism. A total of 82 alleles were detected in the SO cattle and 117 alleles were detected in the PO cattle. The TGLA227 and ETH225 were specific locus candidates which are different in the size and the number of alleles in the SO and PO cattle breeds. The B (HinfI), D (HaeIII), and Y (XbaI) haplotypes were found only in the PO cattle breed samples. The X haplotype was found in all samples of the SO cattle breed but was not found in all samples of the PO cattle breed. The Y haplotype was found in all samples of the PO cattle breed but was not found in all samples of the SO cattle breed. It can be concluded that the TGLA227 and ETH225 (based on microsatellite markers) and the B, D, X, and Y haplotypes (based on the mitochondrial DNA) can be considered as the differentiation factors between the SO and PO cattle breeds.


Introduction
Sumba Ongole (SO) are one of the local Indonesian cattle breeds.The existence of the SO cattle in Indonesia began since the Indian Ongole breed was imported from India in 1914 and centralized in Sumba Island (East Nusa Tenggara Province) for breeding programs (Ministry of Agriculture of the Republic of Indonesia, 2014).Since then, the Ongole generations resulted from breeding programs in the Sumba Island have been known as Sumba Ongole (SO) cattle (Hardjosubroto, 2004).The SO cattle have excellent potential to gain higher dressing percentage (>50%) compared with other local cattle breeds in Indonesia (Agung et al., 2015).
The phenotype characteristics of the SO cattle are closely similar with the Ongole Grade's cattle (known as PO cattle and spread out across in the Java Island), and it is difficult to identify these two Ongole breeds based on the phenotypic parameters because the PO cattle is a crossbred of uncontrolled mating of the SO cattle breed and Java cattle breed (Suyadi et al., 2014) or other Indonesian local breeds (Sudrajad and Subiharta, 2012).In order to resolve the difficulties in the SO and PO identification, a scientific investigation is needed to find the differences between the SO and PO cattle breeds based on their genetic information.
The development of molecular genetic analysis has made it possible to study the potency of certain cattle breeds at the deoxyribonucleic acid (DNA) level.Microsatellites are defined as sequential repeats of a 1-6 nucleotide motif and found throughout the genomes of prokaryotes and eukaryotes (Haasl and Payseur, 2012).The microsatellite markers can be used for parentage verification (Radko, 2010), paternity testing (Stevanovic et al., 2010), assessing the genetic diversity (Seo et al., 2017), and also can be used for estimating the genetic differentiation (Rutledge et al., 2010).
Mitochondria have been characterized as the powerhouses of the cell, because their most basic function is oxidative phosphorylation (Ladoukakis and Zouros, 2017).Mammalian mitochondrial DNA is a gene-dense, doublestranded DNA (dsDNA) molecule of 16.6 kb, which encodes 11 messenger RNAs (mRNAs) (translated to 13 proteins), 2 ribosomal RNAs (rRNAs) (12S and 16S rRNA), and 22 tRNAs (Gustafsson et al., 2016).The mitochondrial DNA can be used for investigation the genetic diversity and genetic structure in certain animal breeds (Sharma et al., 2015).The cytochrome b (Cyt b) gene is one of genes that are located in the mitochondrial DNA (Stewart and Chinnery, 2015) and can be used to investigate the origin of certain animal species (Satish et al., 2009;Zarringhabaie et al., 2011;Farag et al., 2015).The differentiation factor of Indonesian Ongole cattle breeds (SO and PO) might be found based on the microsatellite markers or the Cyt b gene due to the microsatellite markers can be used to identifying the relationship among livestock breeds (Maretto et al., 2012) and the variation in the Cyt b gene can be used for the comparison study of different animal species (Munira et al., 2016).This research was conducted to identify the differentiation factor of Indonesian Ongole cattle breeds (SO and PO) based on 12 microsatellite markers and Cyt b gene polymorphisms.

Blood sample and DNA collection
A total of 50 heads of cattle including the SO cattle (n=25; all individual cattle samples belonged to several private farmers in the Sumba Island) and the PO cattle (n=25; all individual cattle samples belonged to Research Center for Biotechnology farm in West Java) were used for the blood sampling purpose.Blood samples (3-5 mL) were taken from the coccygeal veins using Venoject and collected in Vaccutainer tubes containing an anticoagulant.The blood samples were used in the DNA extraction process using the Genomic DNA Mini kit (Geneaid Biotech Ltd., Taiwan) following the manufacturer's protocol.A total of 12 microsatellite-labeled primers (part of the 30 primers recommended by Food and Agriculture Organization of the United Nations (FAO)) were used in the polymerase chain reaction (PCR) process (primers sequence, annealing temperature, the range of PCR product size, and label used were based on Agung et al. (2015)).Amplification of the Cyt b gene was performed using primers based on Hartatik et al. (2015) i.e. forward (5'-aaaaaccaccgttgttattcaacta-3') and reverse (5'-gcccctcagaatgatatttgtcctca-3').

DNA amplification
The PCR reagents are composed of: KAPA2G Robust Hot Start Ready Mix PCR Kit (Kapa Biosystems, Cape Town, South Africa), forward and reverse primers (200 ng/μL), DNA samples (5-50 ng/μL), and H2O up to 25 μL final volume.The PCR program was set as follows: denaturation at 94°C for 5 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing 58°C to 64°C (depending on the primers) for 45 seconds, extension at 72°C for 45 seconds; and a final extension at 72°C for 5 minutes on Mastercycler® Gradient (Eppendorf, Hamburg, Germany).The PCR products were then visualised by electrophoresis process (1% agarose gel, SyBr® staining, and captured in GBOX documentation System (Syngene, UK)).

Allele and haplotype identification
Multiplex DNA fragment analysis was conducted afterwards for allele identification.The multiplex DNA fragment analysis was conducted in the 1st BASE Laboratory, Malaysia.The haplotype identification was conducted using restriction fragment length polymorphism (RFLP) analysis with three restriction enzymes i.e.HinfI, HaeIII, and XbaI.The use of HinfI, HaeIII, and XbaI enzymes in this study were based on Hartatik et al. (2015), Farag et al. (2015), and Mohamad et al. (2009) respectively.The reagents for RFLP analysis were composed of: 3 µl PCR products, 1.4 µl H2O, and 1 unit (± 0,6 µl) of restriction enzyme HinfI, HaeIII, or XbaI including its buffer (New England Biolabs, USA).The reagents were incubated at 37°C for 1 hour, and it was followed by electrophoresis process.The restriction sites were as follows: G|ANTC, GG|CC, and T|CTAGA for HinfI, HaeIII, and XbaI enzymes respectively.

Data analysis
Data of allele's size (unit in base pairs) were generated using the multiplex DNA fragment analysis.The data was processed using CONVERT version 1.3.1 (Glaubitz, 2004) to convert the size of alleles observed for each individual sample to assure suitability for further data analysis.The converted data was processed using POPGEN version 1:32 program (Yeh and Boyle, 1997) to generate observed number of alleles (nA), effective number of alleles (ne), observed heterozygosity value (Ho), expected heterozygosity value (He), and allele frequency.The converted data was also processed using CERVUS version 3.0.7 program (Kalinowski et al., 2007) to obtain the polymorphism information content (PIC) value.The haplotypes data were generated using PCR-RFLP analysis with three restriction enzymes.The PCR-RFLP products were visualised by electrophoresis process.Individual cattle haplotype was determined based on the differences in the number and size of the visualised bands.The frequency of the haplotype was calculated using MS Excel 2007 program based on Nei and Kumar (2000): χii = (nii/N) for haplotype frequency, where: χii = frequency of ii th haplotype; nii = number of individuals with ii haplotype; N = number of samples.

Result and Discussion
Based on allele identification using multiplex DNA fragment analysis, twelve microsatellite loci in this study revealed high polymorphism, and 199 alleles were detected with 82 alleles in the SO cattle and 117 alleles in the PO cattle.Based on the allele distribution (Table 1  and Table 2), the TGLA122 locus has the highest nA value in the SO cattle while the TGLA122 and  ETH225 were the loci with highest nA value in the PO cattle.The TGLA227 locus in the SO cattle breed has an even-numbered allele size characteristic (e.g.alleles 78, 80, etc.), whereas in the PO cattle breed it was odd-numbered (e.g. 71, 77, etc.).In contrast, the ETH225 locus has an odd-numbered allele size (e.g.alleles 135, 139, etc.) in the SO cattle breed, but it has even-numbered allele size (e.g.alleles 128, 134, etc.) in the PO cattle breed.These results were in agreement with Agung et al. (2015), who reported the odd-numbered alleles in the ETH225 locus and the even-numbered alleles in the TGLA227 locus.However, due to the limited number of samples in this study, a further investigation using a great number of samples for each cattle breed that represents the population of the SO and PO cattle breeds in Indonesia needs to be conducted.
Based on the alleles variation found in the SO and PO cattle, there were several specific locus or allele candidates.The TGLA227 and ETH225 were specific locus candidates which are different in the size and the number of alleles in the SO and PO cattle breeds.This is an indication that the TGLA227 and ETH225 loci might be used to separate the SO and PO cattle breeds.The specific locus in certain cattle breeds was also reported in the Simmental cattle breed.The TGLA53 allele 168 was a specific allele candidate for the Simmental purebred cattle, and the TGLA122 allele 181 was a specific allele candidate for the Simmental crossbred (Agung et al., 2016).
The highest Ho value in the SO cattle breed population was 1.00 (SPS113) and the lowest was 0.16 (BM1818).Meanwhile, the highest Ho value in the PO cattle breed population was 0.92 (SPS113) and the lowest was 0.44 (ILSTS006) (Table 3).
The Ho value can be used for detecting the level of genetic diversity and inbreeding process within a population (Cervini et al., 2006).Unfortunately, the 12 microsatellite markers in the SO cattle and PO cattle in this study mostly were have low Ho value and can be interpreted that the level of genetic diversity was low.However, the high level of genetic diversity in the SO and PO cattle population were represented by TGLA122, ETH225, and SPS113 loci that have high Ho value.
The TGLA122 locus has the highest PIC value in SO cattle (PIC=0.81)and PO cattle (PIC=0.87).Meanwhile, the lowest PIC value in the SO cattle was 0.30 (BM1818) and in the PO cattle was 0.63 (CSSM66).The PIC value at 12 microsatellite loci in the PO cattle breed population in this study was more than 0.5 (PIC>0.5).Hence, every locus in this study was highly informative for detecting the level of genetic diversity in the PO cattle population.Meanwhile, there were four microsatellite loci in the SO cattle breed that have the PIC value less than 0.5 (PIC<0.5).As the result, not every locus in this study can be used to detect the level of genetic diversity in the SO cattle population.In addition, Czerneková et al. (2006) reported that low PIC value can be interpreted that certain conservation process has been carried out in a particular population.
Compared with the results from other studies that also used microsatellites which were mostly identical with our study, some differences can be observed.The differences may be in the minimum and maximum sizes of allele, the number of observed alleles, and also the PIC values.The Ho and PIC values in the SO cattle breed in this study for TGLA53, TGLA227, and BM1818 loci were low.This condition was the same with several of Bos indicus cattle i.e. the  Hissar cattle (Rehman and Khan, 2009), Nellore cattle (Cervini et al., 2006), and Punganur cattle (Kesvulu et al., 2009) 2009).In consequence, the TGLA53, TGLA227, and BM1818 loci were not suitable to investigate the genetic diversity in the SO cattle population.However, the TGLA227 locus was specific locus candidate that might be used to separate the SO and PO cattle breeds.
Based on the mitochondrial DNA analysis results, the size of the PCR product is about 464 base pairs (bp) and the same with the size reported by Hartatik et al. (2015).The haplotypes of the mitochondrial DNA were identified based on the differences in size and the number of bands (RFLP product) that appear in the visualisation process.There were two haplotypes for each restriction enzyme (HinfI, HaeIII, or XbaI) in the SO and PO cattle breed population based on the RFLP analysis.The A and B haplotypes were detected using HinfI enzyme (Figure 1), the C and D haplotypes were detected using HaeIII enzyme (Figure 2), and the X and Y haplotypes were detected using XbaI enzyme (Figure 3).Based on the haplotype data of the mitochondrial DNA (Table 4), the B (HinfI), D (HaeIII), and Y (XbaI) haplotypes were found only in the PO cattle breed samples.Meanwhile, the X (XbaI) haplotype was found only in the SO cattle breed samples.According to the frequency value of the X and Y haplotypes in the SO and PO cattle breeds, these haplotypes were very potential to become a differentiation factor between the SO and PO cattle breeds.

Conclusions
It can be concluded that the TGLA227 and ETH225 loci (based on microsatellite markers) and the B, D, X, and Y haplotypes (based on the mitochondrial DNA) can be considered as the differentiation factors between the SO and PO cattle breeds.

Figure 1 .
Figure 1.The haplotype visualisation based on the RFLP analysis using the HinfI enzyme (M=100 bp ladder size standard; bp=base pair; A=A haplotype; B=B haplotype).

Figure 2 .
Figure 2. The haplotype visualisation based on the RFLP analysis using the HaeIII enzyme (M=100 bp ladder size standard; bp=base pair; C=C haplotype; D=D haplotype).

Figure 3 .
Figure 3.The haplotype visualisation based on the RFLP analysis using the XbaI enzyme (M=100 bp ladder size standard; bp=base pair; X=X haplotype; Y=Y haplotype).

Table 1 .
Alleles distribution and its frequency based on 12 microsatellite loci in the SO cattle breed bold=the alleles that was not found in the PO cattle breed.

Table 2 .
Alleles distribution and its frequency based on 12 microsatellite loci in the PO cattle breed bold=the alleles that was not found in the SO cattle breed.

Table 3 .
Characterization of the twelve microsatellite markers in the SO and PO cattle breeds

Table 4 .
The haplotypes frequency in the SO and PO cattle breeds