Polymorphism of Growth Hormone (GH) Gene in Lakor Goat from Lakor Island of Southwest Maluku Regency

Lakor goat survive in Lakor island in Southwest Maluku with high temperature and limited water. Growth trait in goat is interest to explore cause related with economic trait that encoded by growth hormone (GH) gene. The aim of this study was identify of polymorphism GH gene of Lakor goat in Lakor island. A total of 63 samples were collected from three locations (village) i.e Ketti Letpey (18), Werwawan-Yamluli (26), and Letoda (19). DNA was extracted from hair follicles. A 422 bp specific DNA fragment was successfully amplified and genotyped by PCR-RFLP method using HaeIII enzyme. Results showed that polymorphism was found with two variant of genotypes (AA and AB) and two alleles (A and B). AB genotype was dominant in all of populations (93.7%) with A and B alleles were 0.53 and 0.47, respectively. Heterozygosity observed and expected value reached 0.502 and 0.498, respectively while Polymorphic Information Content was in moderate values (0.374). All of populations were in disequilibrium genetic. It maybe caused limited buck and nonrandom mating in population that effect of low genetic variation. Inbreeding study are needed to explore it. The introgression of bucks from other families in several locations within Lakor island can be an alternative solution to increase the genetic diversity of the lakor goat population.


Introduction
Goat is one of the livestock of the principal meat producing animals in Indonesia and has immense contribution to the resource poor section of society for their livelihood. Several goat breeds that have adapted to the environment and geography in the territory of Indonesia, they are Marica, Bengal, Etawah, Kosta, Kacang, Gembrong, Muara, Samosir (Batubara et al., 2013), and Lakor goats (Volkandari et al. 2019;Rumanta et al. 2020). Budisatria et al. (2008), stated that goats are known as livestock that produce meat, milk, and fur, but are also used in traditional and religious ceremonies. Lakor goat is a domestic goat (Capra hircus) have been successfully bred and propagated in habitats with high temperature, limited rainfall and forage, but have very good adaptability. They represent a unique genetic resources by virtue of their adaptability, resistance to many infectious diseases and prolificacy in the tropics of Indonesia. Lakor goat has protected based on the Minister of Agriculture Decree No.
2912/Kpts/OT.140/6/2011 with a very limited distribution, only on Lakor Island. Salamena et al. (2014) reported that, Lakor goat is suspected to be a cross-breed between the Etawah goat descendant and the Kacang goat (Indonesian native goat), in which genetically Etawah is dominant. Based on morphological characters, color of Lakor goat is varies from white, black, brown, grey, crème, and combination of those five colors. Volkandari et al. (2019), reported that body color of Lakor goat has a combination of solid color with black and white, grey, brown, and spot black white while head color is dominant of black. Growth hormone (GH) plays a vital role in different biological processes, such as growth, metabolism, lactation, and reproduction of livestock species (Katoh et al. 2008;Seevagan et al. 2015). Physiologically, GH is released from the anterior lobe of pituitary gland, where its main effects are associated with the stimulation of growth of bones and skeletal muscles, through the action of insulin-like growth factor (IGF-1) (An et al. 2011), as well as its great role in milk production (Akers, 2006). Lakor goats have been Buletin Peternakan 44 (4): 194-199, November 2020 Bulletin of Animal Science ISSN-0126-4400/E-ISSN-2407-876X Accredited: 36a/E/KPT/2016 http://buletinpeternakan.fapet.ugm.ac.id/ successfully bred and propagated in habitats with high temperature, limited rainfall and forage, but have very good adaptability. They represent a unique genetic resources by virtue of their adaptability, resistance to many infectious diseases and prolificacy in the tropics of Indonesia.
Livestock exhibiting high genetic merit in growth and body measurements receive high priority inbreeding programmers for meat purpose (Susilorini et al. 2017). Although, lot of progress has been achieved in animal improvement using conventional breeding methods, environmental influences limits accuracy of such methods for improving polygenic inheritance like body measurements (Bin et al. 2009). However, the genetic improvement of such traits can be enhanced by marker assisted selection, which is highly accurate in estimating breeding value of livestock (Dekkers, 2004). In view of the pivotal role of growth hormone in livestock growth and development, GH gene may be used as a candidate gene for studying its polymorphism and association in relation to growth. Supakorn (2009), asserted that GH gene may be used as a candidate gene for studying its polymorphism and association in relation to growth.
Recently, genetic polymorphism in candidate genes has affecting role in production traits, have encouraged considerable research interest due to their ability for utilization as an aid to genetic determination and to demarcate evolutionary relationships in different livestock breeds (Sodhi et al., 2007). Polymerase Chain Reaction'' (PCR) and Restriction Fragment Length Polymorphism'' (RFLP), assist to study the specified genes and to amplify them in vitro (Liu and Cordes, 2004). Many researchers studied an association of polymorphism of growth hormone gene with body weight and growth like. Genetic identification of the lakor goat population based on the identification of the GH gene polymorphism is very important. This Information in can be used to early information of their status and genetic diversity. The present study was undertaken to identify GH gene polymorphism of Lakor goat in Lakor island from Southwest Maluku Regency. The results of this study will serve as scientific information for the government of Southwest Maluku regency regarding the conservation of native livestock in the future.

Ethical approval
This study does not require ethical approval because using non-invasive samples (hair follicles)

Samples and DNA extraction
A total of 63 samples of Lakor goat

Genotyping Procedure
Genotyping of Growth Hormone (GH) gene used PCR-RFLP method. GH gene was amplified by a pair primer from Hua et al. (2009), Forward: 5'-CTCTGCCTGCCCTGGACT-3' and Reverse: 5'-GGAGAAGCAGAAGGCAACC-3'. Total volume of mixture was 10μl containing of 5μl PCR Mastermix (Bioline, Biosystems), 1μl (10 pmol/μl) of each primer, 2μl water free nuclease and 1μl DNA template. PCR mixture was run by thermal cycler machine (Techne Plus, United State) following program: pre-denaturation at 94 o C for 5 minutes, 35 cycles of 95 o C for 30 seconds, 65 o C for 30 seconds, 72 o C for 45 seconds and a final extension at 72 o C for 7 minutes (Hua et al., 2009). PCR products were checked using 1% agarose gel (100V, 1 hour) and then stained by Ethidium Bromide. Gel were visualized under UV light (UV Transimullator, USA) and taken of picture by camera.
For genotyping, HaeIII restriction enzyme was used. PCR products were digested overnight at 37 o C in waterbath. Fifteen microliter of RFLP mixture was prepared containing 3μl of PCR product, 1.5μl buffer tango, 0.3μl (10U/μl) of HaeIII enzyme, and 10.2μl water free nuclease. RFLP product was electrophoresed by 2% agarose gel and run with 100 Voltage for 1 hour. Gel was visualized under UV light (UV Transimullator, USA) and taken of picture by camera. Genotype of each samples were identified using genotype patterns from Hua et al. (2009).

Data analysis
Genotype and allele frequencies, heterozygosity observed and expected, polymorphic information content (PIC) were analyzed by GenAlex 7.0 (Kalinowski et al. 2007) while Hardy Weinberg Equilibrium (HWE) was directly calculated by Nei and Kumar (2000).

Results and Discussion
A 422 bp specific target of Growth Hormone (GH) gene was successfully amplified in all of samples ( Figure 2). The DNA fragment was spanned from 727 to 1148 according GenBank Accession No. D00476.1 where located at exons 2 and 3. Transition mutation at 781 Adenine to Guanine (A781G) that caused amino acid change from Serine to Glycine at the residue 35. The mutation was recognized by HaeIII (GG^CC) restriction enzyme (Figure 3). Three genotype patterns was detected i.e AA, AB and BB with two alleles (A and B). Genotype AA was detected with 366 and 56 bp; AB genotype is 422, 366 and 56 bp while BB genotype is 422 bp (Hua et al. 2009). In this study, only two genotypes were found, AA and AB (Figure 4) while BB genotype was absence.
Genotype AB of Lakor goat was dominant in all of population (Ketti Letpey, Werwawan Jamluli and Letoda, 94.4%; 92.3% and 94.7%, respectively). Frequency of A allele higher than B (Table 1). The result of this study has similar with previous studies, in Chengdu-Ma goat from China (Bai et al. 2005), Boer goat (Hua et al. 2009), Savana and Kalahari goat from Malaysia (Marini et al. 2012), Kacang goat from Indonesia (Ilham et al. 2016), and Saburai goat from Indonesia (Sulastri et al., 2017). Conversely to the other studies, Mahrous et al. (2018), reported that BB genotype was detected 5-10% and absence of AA genotype of three breed goat (Barki, Damascus and Zaraibi) from Egypt. In India, Barbari and Sirohi goat were detected AB (82% and 90%, respectively) and BB (18% and 10%, respectively) genotypes (Singh et al. 2015). These data suggest that there has been a significant amount of inbreeding depression within populations. This is supported by the fact that buck with strong genetic quality have not been introduced to Lakor Island, so the breeding process that occurs in the population takes place naturally and has the potential to result in inbreeding depression.
Polymorphic Information Content (PIC) value in this study was 0.374 that mean was reasonably informative. Bostein et al. (1980), classified of PIC value became 3 groups, slightly  (0-0.25), reasonably (0.25-0.5), and highly (>0.5) informative. Guo and Elston (1999), PIC value is often used to measure the informativeness of genetic markers. Loci with many alleles and PIC near 1 are most desirable (Bostein et al. 1980). Based on analysis, all of the populations of Lakor goat were in disequilibrium genetic (HWE).
Equilibrium genetic in population can be disturbed of several factor i.e. number of forces, nonrandom mating, mutation, natural selection, genetic drift, introgression, and gene flow (Falconer and Mackay 1996). Limited of buck in Lakor island may be cause of low genetic diversity that proven with heterozygosity observed (Ho) value higher Forward Reverse  (Keller and Waller 2002;Hardjosubroto 2004;Charlesworth and Willis 2009). For decreasing of inbreeding rate, we suggest two alternatives: 1) identify of inbreeding rate is needed; and 2) rotation of buck from other locations or village in Lakor island.
Research by Volkandari et al. (2019) and Rumanta et al. (2020) reported that there is low genetic variation within the Lakor goat population after few generations of selection and domestication. This condition is due to improper breeding mechanisms and broodstock management. Conventional breeding practices lead to a loss of genetic where the contribution of parent is practically unknown. Moreover, this practice makes it difficult for selection of the next generation, where the relationships and genetic lineages of the resulting offspring are unclear. This will lead to inbreeding which, in turn, contributes to a loss of genetic variation within the Lakor goat population. In this study, we have found that genetic variation in populations is reduced, and it is important to protecting genetic loss so that it is not significant.
Currently, the policy being socialized related to the use of genetic information from the GH gene to farmers on Lakor island is controlling breeding mechanisms and broodstock management may be done by selecting buck from neighboring villages and not from the same village as doe. Rumanta et al. (2020), reported that on Lakor Island, respective villages have a permanent fence composed of rocks (local name is Lutur) to protect the mixing of livestock between villages. We assume that, in this way, the rate of genetic loss in the Lakor goat population can be reduced. This is very important for the traditional livestock industry on Lakor Island and to maintain the future sustainability of this endemic species. If no mitigation measures are taken, the loss of genetic variation in the population may result in severe effects in the future.

Conclusions
This research found the highly significant Chi-square values for Lakor goat breed revealed that not was in Hardy-Weinberg equilibrium for this region of GH gene. Results showed that polymorphism was found with two variant of genotypes (AA and AB) and two alleles (A and B). AB genotype was dominant in all of populations (93.7%) with A and B alleles were 0.53 and 0.47, respectively. We asserted that The Lakor goat population has a high level of inbreeding depression as a result of geographical isolation and not have been the introduction of buck from other breeds. However, as the study was based on a limited sample size, further study on larger sample is suggested.