Genetic Polymorphism of Local Goat Gorontalo Indonesian Based on Microsatellite Markers

The goat population in Southeast Asia in 2019 was 37,844,420 heads, and 50.14% (18,975,955 heads) came from Indonesia (Food and Agriculture, 2020). Local goats in Indonesia exhibit varying characteristics due to differences in geographic location and nurture systems among farmers. Gorontalo, located on Sulawesi Island in central Indonesia, is home to local goats i.e. local Gorontalo goat that have thrived in the environment and farming culture (Ilham et al., 2016; Kunuti et al., 2021). Gorontalo, along with five other provinces on the island of Sulawesi, have the potential to become an area producing animal products because, apart from being located in the middle of Indonesia, it is also close to the Philippines for livestock marketing purposes. Based on data from Badan Pusat Statistik (BPS) (2025), the total goat population in Gorontalo in 2024 was recorded at 17,780 heads, representing approximately 0.11% of Indonesia’s total goat population of 15,710,055. Furthermore, Gorontalo province is inhabited by two other goat breeds, namely Kacang and Etawah Crossbreed, with the local Gorontalo goats believed to be a crossbreed between the two. The Kacang goat is a native breed of Indonesia, similar to local goats in Malaysia, Thailand, Myanmar, and the Philippines (Pralomkarn et al., 2012; Tsukahara et al., 2008), while the Etawah Crossbreed is the result of crossbreeding the Etawah goat and the Kacang goat (Suyadi et al., 2021). Based on body weight, shoulder height, body length, ear length, and ear width, local Gorontalo goat are different from Peranakan Etawah goat and Kacang goat (Ilham et al., 2023), but it needs to be confirmed through molecular approaches.

Examinations on the potential and genetic polymorphism of local goats across several Indonesian islands, including local Gorontalo goat are very limited (Ciptadi et al., 2018). Genetic polymorphism of the Growth Hormone (GH) gene (Ilham et al., 2016) and the IGF-1 gene (Ilham et al., 2016) on Kacang goat in Gorontalo were polymorphic but the heterozygosity value was low. This low value of heterozygosity can affect the genes that controls the immune system as well as increasing the inbreeding pressure in certain population. Consequently, it decreases individual adaptability and survival to environmental changes, which can increase the risk of extinction (Radwan et al., 2010). Genetic polymorphism and distance in goats can be determined using genetic markers such as Simple Sequence Repeats (SSR)/Short Tandem Repeats (STR)/Simple Sequence Length Polymorphisms (SSLP)/Variable Number Tandem Repeats (VNTR)/ Microsatellite (Guichoux et al., 2011; Hadi et al., 2020; Vieira et al., 2016). Genetic markers measure genetic diversity and distance within and between populations. The greater the genetic distance, the higher the population’s genetic diversity.

A microsatellite is a DNA sequence consisting of 4 DNA bases (A, G, T, C) with short repeat motifs, found in many eukaryotic chromosomes (Al-Khalaf et al., 2022). Microsatellite DNA offers several advantages which include its abundance in the genome, naturally co-dominant, and high polymorphism, with low DNA template requirements (10-100 ng) (Bennett, 2000; Raza et al., 2016). The use of microsatellite DNA as a genetic marker in local livestock in Indonesia has been widely applied in local cattle (Agung et al., 2019), goats (Pakpahan et al., 2016; Susilorini et al., 2022), and buffalo (Saputra et al., 2020).

The identification of genetic polymorphism using genetic markers is more accurate compared to phenotype markers, as they are not affected by environmental variations (Brito et al., 2017). Furthermore, genetic characterization using molecular approaches can complement research results based on morphometrics (Singh et al., 2019). Research on phenotype diversity based on qualitative and quantitative traits and genetic diversity based on PCR-RFLP in local goats in Gorontalo started in 2012, but research using microsatellite markers has never been done. This research aims to determine the variaton and genetic distance of three local goat breeds in Gorontalo, Indonesia, based on microsatellite DNA. The novelty of this study is the discovery of genetic variation in 3 microsatellite loci of local Gorontalo goats. This information is additional information on the genetic diversity of local goats in several regions in Indonesia for molecular basis selection. The results of this research are expected to enrich knowledge about the genetic polymorphism of local goats in Indonesia, support the conservation of local livestock diversity, and enhance the effective use of genetic resources.

The blood samples were collected from Gorontalo Province in Indonesia, covering 10 District, namely Dungingi, Kota Barat, Kota Utara, Bulango Timur, Bulango Utara, Kabila, Kabila Bone, Tibawa, Telaga, and Paguyaman (Figure 1). Genomic DNA extraction and targeted DNA amplification were performed at the Animal Biotechnology Laboratory, Brawijaya University (UB) Malang.

A total of 128 blood samples of different goat breeds were used, including 19 Etawah Crossbreed, 87 local goats Gorontalo, and 22 Kacang goats. The blood was collected using venoject needles in vacutainer tubes containing Ethylenediaminetetraacetic acid (EDTA) from the jugular vein, yielding approximately 4 ml, which were stored in -20°C before genomic DNA extraction.

Genomic DNA was extracted from the blood using Cell lysis, Nuclei Lysis, Protein Precipitation, DNA Precipitation, and Rehydration steps based on the protocol of the Wizard Genomic DNA Purification Kit (Promega, USA). The quality of the extracted DNA was checked using agarose gel electrophoresis. The amplification of the three microsatellite DNA loci (Table 1), as recommended by the International Society for Animal Genetics/Food and Agriculture Organization (ISAG/FAO) for goats, was performed using the Polymerase Chain Reaction (PCR) method. The three microsatellite loci in this research to detect the genetic diversity of local Indonesian goats, and the results obtained were highly polymorphic (Susilorini et al., 2022).

The initial step of amplification involved adding 1 μL of the DNA sample into the first tube and temporarily storing it in the refrigerator. Taq DNA Polymerase used is Gotaq green master mix by Promega. The primer concentration used is 10 micromolar, and the DNA template concentration is <250 ng. To prepare the PCR premix, 15 μL of Taq DNA polymerase was mixed with 14.4 μL of distilled water (DW), 0.3 μL of forward primer, and 0.3 μL of reverse primer into the second tube, and vortexed for approximately ±10 seconds. The PCR premix was added to the first tube containing 1 μL of DNA and spun down for approximately ±10 seconds to ensure homogeneity. The mixture was transferred into the thermocycler and the machine was set for the PCR process, starting with an initial denaturation at 95ºC for 5 minutes, followed by 35 cycles of denaturation at 95ºC for 10 seconds, primer annealing for 20 seconds, extension at 72ºC for 60 seconds, and a final extension at 72ºC for 5 minutes. The PCR product (3 μL) was visualized by electrophoresis (Mupid-exU, Japan) for 40 minutes at 100 volts in a 1% agarose gel with Nucleic Acid Dye (Diamond, Promega). The size of the target DNA product was determined using a 100 bp DNA size standard (Promega). Subsequently, the PCR product bands were visualized using a GelDoc (Glite 965 GW, Taiwan), and confirmed microsatellite DNA PCR products were sent to 1Base Laboratory, Selangor, Malaysia for fragment analysis.

Genetic polymorphism analysis after fragmentation involved several steps, namely calculating the number and frequency of alleles, heterozygosity, Polymorphism Information Content (PIC), and fixation indices (Fis, Fit, and Fst) (Nei and Kumar, 2000). Meanwhile, the genetic similarity and distance were calculated based on microsatellite allele frequencies in each population. The phylogenetic reconstruction among local Gorontalo, Etawah Crossbreed, and Kacang was performed using the Unweighted Pair Group Method with the Arithmetic (UPGMA) method, assuming equal evolutionary rates among goat strains/groups. The number and frequency of alleles, heterozygosity, fixation indices, genetic similarity, distance, and the phylogenetic tree were determined using the statistical tool PopGene 1.32. The phylogenetic tree reconstruction was performed using MEGA software version X (Kumar et al., 2018).

The analysis of the three microsatellite DNA loci in local goats in Gorontalo showed that the number of alleles at the OarFCB20, MCM527, and ILSTS011 loci was 5, 11, and 8, respectively, as presented in Table 2 and Figure 3. Based on breed, the average observed allele (Na) in local Gorontalo goats was higher at 7.67±3.06 compared to Etawah Crossbreed at 6.00±2.65 and Kacang at 4.33±2.31, as shown in Table 3. The allele frequency were slightly higher in local Gorontalo goats compared to Etawah Crossbreed and Kacang goats. This is not much different from previous phenotype research, namely that the phenotype of local Gorontalo goats is more varied than that of Etawah Crossbreed and Kacang goats (Ilham et al., 2023). The number of alleles in this research was not significantly different from other research in Indonesia, such as Etawah Crossbreed (4.17±0.72), Kacang (5.67±1.67), Senduro (5.75±1.36) (Susilorini et al., 2022),

Table 1: Three Microsatellite loci, primer sequences, base pair range, annealing temperature, and fluorescent label recommended by ISAG/FAO for assessing genetic polymorphism in the three local goat breeds in Gorontalo, Indonesia

Table 3: Heterozygosity based on microsatellite DNA in Etawah Crossbreed, local Gorontalo, and Kacang goats at Gorontalo, Indonesia

Noted: n: number of samples; Na: number of observed alleles; Ne: number of effective alleles; Ho: observed heterozygosity; He: expected heterozygosity; X2: chi-square test; HW: Hardy-Weinberg; *P<0.05 (significantly different at 0.05)

Jawarandu (4.9±2.2), Kosta (2.5±1.3), Gembrong (2.2±1.1) (Zein et al., 2012). These allele numbers were slightly lower than the Girgentana Siria goat at 8.36±2.69 (Sardina et al., 2015) and the Yunnan China goat at 11.4 (Guang-Xin et al., 2019).

The more alleles in a population indicate the higher diversity. More alleles in local Gorontalo goats indicate higher diversity due to crossbreeding between Peranakan Etawah and Kacang goats. Polymorphism is lower in Kacang goats because the selection process is strict, and breeders do not carry out crossbreeding, so only certain alleles appear to dominate. The proportion of homozygous alleles will increase, the proportion of heterozygotes will decrease, and inbreeding depression will occur if family marriages last long in a population (Rafter et al., 2022).

As shown in Table 2, the highest-frequency alleles at the OarFCB20, MCM527, and ILSTS011 are alleles 100, 156, and 271, with respective frequencies of 0.713, 0.320, and 0.447. Among the different breeds, the Kacang goats exhibited the highest allele frequencies for each observed locus are found in Kacang goats, with values of 0.864 (allele 100), 0.636 (allele 156), and 0.682 (allele 271). Meanwhile, the local Gorontalo goats showed the lowest allele frequencies observed at 0.018 (allele 107), 0.011 (allele 164), and 0.006 (allele 269). A locus can be considered polymorphic when the number of alleles in the population at that locus is more than one or the allele frequency is less than or equal to 0.95 (Nei and Kumar, 2000). Therefore, the polymorphism indication in the local Gorontalo goat population suggested that it can be considered for selection purposes to improve and enhance genetic quality. Selection for traits of high economic value in goats can maximize birth weight and avoid dystocia due to high birth weights (Jawasreh and Al-Kass, 2023).

Four (4) unique/specific alleles in local Gorontalo goats that were not found in Etawah Crossbreed and Kacang goats, namely allele 107, allele 170, and allele 275 and 276 at the OarFCB20, MCM527 and ILSTS011 loci, respectively (Table 2). These unique alleles have the potential to be used as genetic markers to differentiate between two local goats in Gorontalo, Indonesia. Specific or unique alleles can be used for breeding purposes and predicting or distinguishing specific genotypes (Behera et al., 2012; Thudi and Fakrudin, 2011).

The three observed microsatellite loci in Table 2 were considered polymorphic because they had more than 2 alleles. The highest Polymorphism Information Content (PIC) value of 0.73 was found at the MCM527 locus, followed by the ILSTS011 locus at 0.57, and the OarFCB20 locus at 0.45, which was the lowest. The MCM527 locus had the highest average value because the PIC values of each breed were also high (>0.5), which were 0.81, 0.82, and 0.56 for Etawah Crossbreed, local Gorontalo, and Kacang goats, respectively. Microsatellite loci with PIC values >0.5, 0.25-0.5, and <0.25 were considered highly, moderately, and low informative (Asroush et al., 2018; Dixit et al., 2013).

The moderate PIC value at the OarFCB20 locus was due to the lower number of alleles ranging from 3 to 5 in each breed compared to the MCM527 and ILSTS011 loci. The PIC value depends on the number of alleles and their frequency distribution, therefore, the value will be higher when there are more than two alleles or lower allele frequencies (Raza et al., 2016). Research on five local goat breeds in East Java showed that the PIC values for the OarFCB20, MCM527, and ILSTS011 loci were 0.662, 0.681, and 0.667, respectively (Susilorini et al., 2022). The PIC value for the ILSTS011 locus in Yunnan China goats was 0.505, and for the OarFCB20 locus was 0.636 (Guang-Xin et al., 2019). In Beetal goats from Pakistan, the PIC values for the OarFCB20, MCM527, and ILSTS011 loci were 0.709, 0.668, and 0.38, respectively (Naqvi et al., 2017). PIC measures the ability of a marker to detect polymorphisms; therefore, the selection of markers for genetic studies is essential (Serrote et al., 2020). Meanwhile, the high average PIC values in this research indicated that the MCM527 and ILSTS011 loci can be recommended as markers of selection for Etawah Crossbreed, local Gorontalo, and Kacang goats in Indonesia. Using selection methods based on molecular information can shorten the selection period and achieve genetic improvement more quickly (Khdir et al., 2023).

As shown in Table 3, the average observed heterozygosity (Ho) in local Gorontalo, Kacang, and Etawah Crossbreed goats, was 0.48, 0.42, and 0.40, respectively. The highest heterozygosity in Etawah Crossbreed and local Gorontalo goats was found at the MCM527 locus, with values of 0.74 for both breeds, while the ILSTS011 locus had 0.55 in Kacang goats. Meanwhile, the lowest heterozygosity in Etawah Crossbreed, local Gorontalo, and Kacang goats were observed at the OarFCB20 locus, with values of 0.05, 0.17, and 0.27, respectively. The lower observed heterozygosity at the OarFCB20 locus was due to the lower number of alleles. Previous research had shown that the observed heterozygosity in Etawah Crossbreed, Kacang, and Senduro was 0.592±0.211, 0.470±0.135, and 0.479±0.178 (Susilorini et al., 2022), while in Jawarandu, Gembrong, and Kosta had values of 0.463 ± 0.207, 0.372 ± 0.173, and 0.395 ± 0.214 (Zein et al., 2012). The observed heterozygosity (Ho) in native Brazilian goats was 0.672 (Menezes et al., 2006), while in Baladi, Zaraibi, Demusces, and Farafra goats in Egypt, the values were 0.155, 0.211, 0.100, and 0.222, respectively (El-Sayed et al., 2017). Heterozygosity is a measure of genetic diversity within animal populations, where a higher number of detected alleles in a locus leads to greater heterozygosity values. A heterozygosity value approaching one indicates increasing genetic diversity in the population, while a value approaching zero indicates reduced genetic variation (Knapp, 2018).

The average observed heterozygosity (Ho) in Etawah Crossbreed goats was lower than the expected heterozygosity (He) (0.40<0.71), as well as in local Gorontalo goats (0.48<0.63). In Kacang goats, the Ho was higher than the He, with a value of 0.42>0.43, however, there was no significant difference between the values, as presented in Table 3. The imbalance between observed and expected heterozygosity indicated the occurrence of random mating among individuals within the population. When the observed heterozygosity was lower, it indicated that the inbreeding occurred within the population, while higher values suggested a relatively large diversity (Asroush et al., 2018). Moreover, mating patterns and migration had been established as factors that can affect the level of heterozygosity in a population. Low genetic diversity can occur in a livestock group due to the limited introduction of new males into the population, while high diversity indicated the absence of intensive selection programs or inbreeding pressure (Jakaria et al., 2012).

Except for the OarFCjB20 locus in Kacang goats, all microsatellite loci in the three local goat breeds observed did not show the Hardy-Weinberg equilibrium condition (Table 3). Genetic imbalance in each local goat population illustrates low heterozygosity and unequal distribution of alleles. Factors thought to influence are selection by breeders and inbreeding. Randomly combining gametes through the mating process causes genotype and allele frequencies to remain constant from generation to generation. Hardy-Weinberg equilibrium can be achieved if the assumptions are met that mating occurs randomly, there is no natural selection, the population size is very large, no genetic drift, no mutations occur, and the locus is autosomal (Lachance, 2016). It will always be difficult for a livestock population to achieve a condition of genetic balance in the real world because breeders are always involved in selecting and mating their livestock.

The average values of Fis at 0.291 for the three observed loci were lower compared to the value of Fit at 0.360 but higher than Fst at 0.102, as shown in Table 4. The locus that contributed the most to the average fixation index components (Fis, Fit, Fst) was OarFCB20, followed by ILSTS011 and MCM527. The values of the fixation index in this research indicated higher genetic polymorphism among individuals within the population (Fit) in the three local goat breeds in Gorontalo compared to within breed/subpopulation (Fis) and among breed/subpopulation (Fst). Furthermore, the fixation index of the three local goat breeds in Gorontalo is higher than that of the five local goat breeds in East Java, where Fis, Fit, and Fst was 0.258, 0.324, and 0.088 (Susilorini et al., 2022), and in seven indigenous Vietnamese goats with Fst, Fit, Fis of 0.083, 0.178, and 0.104, respectively (Thuy et al., 2017).

Table 4: Values of fis, fit, and fst (fixation index) for the three microsatellite DNA loci in Etawah crossbreed, local Gorontalo, and Kacang goats at Gorontalo, Indonesia

The F-statistic or fixation index reflected selection and mating patterns, as well as their relationship to polymorphic alleles. A negative Fis value indicated relatively high heterozygosity, while a positive value showed high homozygosity (El-Sayed et al., 2017; Tefiel et al., 2018). Moreover, populations with low genetic diversity were more likely to consist of closely related individuals, leading to a higher chance of inbreeding within the group. Based on previous research, Fis values indicated inbreeding within the population, Fit showed inbreeding within a subpopulation, and Fst indicated genetic differentiation between populations (Zein et al., 2012). An inbreeding coefficient above 12.5% indicated significant inbreeding pressure within the population. The Fis values in Table 4 showed the presence of inbreeding within the subpopulations of Etawah Crossbreed, local Gorontalo, and Kacang goats. The high inbreeding values were attributed to small population size, a limited number of males, and a restricted distribution area (Brito et al., 2017; Rafter et al., 2022). Inbreeding causes reduced reproductive quality, increased susceptibility to pathogens (Wright et al., 2008), and decreased survival ability in the wild (Halverson et al., 2006). Goats quality and reproductive efficiency can be evaluated based on the aspects of fertility, conception, kidding, productivity, and twinning rates (Alkass et al., 2021).

The genetic distance between local Gorontalo and Kacang goats was 0.078, which was closer than the distance between Etawah Crossbreed at 0.164 as well as Etawah Crossbreed and Kacang goats at 0.452, as illustrated in Table 5. The genetic distance indicated the genetic difference between species or populations within a specific species. Therefore, smaller genetic distance values approaching zero indicated a closer genetic relationship between two or more individuals, while larger values approaching one indicate a more distant relationship (Jiyanto et al., 2014). The genetic distance between the three local goats in Gorontalo was higher compared to the distance between Etawah Crossbreed and Kacang goats in East Java, Indonesia at 0.077, Etawah Crossbreed and Senduro goats at 0.037, as well as Kacang and Senduro goats at 0.055 (Susilorini et al., 2022). Proximity in the genetic distance indicated a high genetic similarity due to shared ancestry (Ofori et al., 2021).

Table 5: Nei’s genetic identity (above diagonal) and genetic distance (below diagonal) between Etawah crossbreed, local Gorontalo, and Kacang goats

The genetic distance values used to construct the phylogeny of the three breeds as shown in Figure 2, indicated that local Gorontalo goats were grouped in the same cluster as Kacang, while Etawah Crossbreed formed a separate cluster. This phylogeny also showed that local Gorontalo goats were more similar to Kacang goats compared to Etawah Crossbreed. Even though microsatellite DNA is genetically closer, research on average body weight and several body measurements by Ilham et al. (2023) on local Gorontalo goats previously showed higher values than Kacang goats. This closeness was due to the higher frequency of mating between local Gorontalo and Kacang goats compared to Etawah Crossbreed, resulting in the genetic proportion of Kacang becoming more dominant in local Gorontalo goats. Pakpahan et al. (2016) stated that most local goats in Indonesia showed a closer relationship with Capra aegagrus than Capra falconeri. The genetic distance between the local goats and Capra hircus was 0.003-0.004, which was closer compared to Capra falconeri and Capra aegagrus (Suyadi et al., 2022). Furthermore, local Indonesian goats based on Kacang goats showed a closer genetic distance and formed a single cluster compared to Gembrong goats (Zein et al., 2012). This research also supported previous research that Kacang goats were the main genetic source of all current local goat breeds in Indonesia (Suyadi et al., 2019).

The results of this study are important in practical applications in the field of animal husbandry, including providing a clear direction in conservation and breeding efforts based on genetic data. Strengthening the position of the Local Gorontalo goat as a unique plasma nutfah that needs to be maintained. Supporting national policies in protecting local livestock genetic resources.

This study confirmed that the three goat breeds in Gorontalo (Etawah Crossbreed, Local Gorontalo, and Kacang goat) are genetically diverse and polymorphic based on microsatellite markers. The Local Gorontalo goat showed closer genetic proximity to the Kacang goat than to the Etawah Crossbreed. The microsatellite loci OarFCB20, MCM527, and ILSTS011 proved to be informative markers for assessing genetic diversity. These findings provide valuable insights for genetic conservation and selection strategies of local goats in Indonesia.

This research fund is supported by the Indonesia Endowment Fund for Education Agency (LPDP) of the Ministry of Finance of the Republic of Indonesia.

Research on phenotype diversity based on qualitative and quantitative traits and genetic diversity based on PCR-RFLP in local goats in Gorontalo has been conducted since 2012. Still, with microsatellite markers, it has never been done. The novelty of this study is the discovery of genetic variation in 3 microsatellite loci of local Gorontalo goats, namely locus OarFCB20, MCM527, and ILSTS011. Apart from diversity, information was also obtained that local Gorontalo goats are closer to the Kacang goat compared to the Etawah Crossbreed goats. This information is additional information about the genetic diversity of local goats in several regions in Indonesia in the context of selection based on molecular information.

FI: Collecting data, doing the research and preparing the manuscript.

GC, TES: Supervision and review the manuscript.

AA: Data analysis.

CDN, PPA: Data analysis, preparing the manuscript.

SS: Conceptualization, supervision, review the manuscript, correspondence.

All authors contributed to the article and approved the submitted version.

The protocol of this study was approved by the Gorontalo State University Health Research Ethics Commission (KEPK) Number 44B/UN47.B7/KE/2023 approved the procedure for using animals in this study.

The authors declare that no Genrative AI was used in the creation of this manuscript.

The authors have declared no conflict of interest.