GENETIC CHARACTERIZATION IN FOUR INDONESIAN CATTLE BREEDS INFERRED FROM ILLUMINA PARENTAGE SNP MARKERS

Genetic characterization in native livestock is essential to conserve the genetics standard of livestock for pure-breeding programs. This research aimed to characterize four Indonesian cattle breeds using 156 sites of Parentage SNP Markers, includingARS-USMARC-Parent (119 sites) and ARS-USMARC (37 sites). Total of 113 mixed-sex animals were used for the evaluation, including 16 Bali ( Bos javanicus ), 16 Madura ( Bos indicus ), 33 Ongole grade ( Bos indicus ), and 48 Sumba Ongole ( Bos indicus ) cattle breeds. Results showed Bali, Madura, and Ongole lineage (Ongole grade and Sumba Ongole) cattle can be discriminated against with the Parentage SNP Markers. The pairwise genetic distance analysis revealed that Bali cattle had a closer genetic relationship with Madura cattle (0.043 - 0.151) than Ongole lineage cattle (0.073 - 0.325). However, the phylogenetic tree explained that the Ongole lineage cattle are grouped into two different clades. At the same time, Bali, Madura and Ongole lineage cattle are grouped into their original cluster. The study suggests that the Parentage SNP Markers can be used to characterize Bali, Madura, and Ongole lineage cattle accurately.

. Recently, the crossbreeding program with exotic bulls was used to increase meat production (44).Consequently, many farmers applied crossbreeding with the frozen sperm of exotic cattle to increase meat production.As a result, the crossbred cows produced from the crossbreeding between exotic bulls (Bos taurus) and native cows have low reproductive performance (17,23,34).Despite reducing the reproductive traits, the crossbreeding between native cows and exotic cattle can influence the phenotypic and physical characteristics (41,3).Consequently, the origin of genetic structure in native cattle will be changed when crossbreeding is not controlled.Hence, genetic characterization helps select purebred cattle for the breeding program.Currently, there are many studies to characterize Indonesian cattle breeds with many regions of mtDNA genes such as Cytb (35,25,42,31).Despite those previous studies, many studies were conducted for genetic characterization in Indonesian cattle breeds with microsatellite markers (1) and SRY gene (17).Currently, the genetic characterization of cattle can be evaluated based on their genomic diversity through Illumina Bovine Beadchip (49,32,26).Many previous studies used the Bovine Beadchip toolkit to characterize native cattle breeds in South Africa (30), China (47), semi-arid areas of Africa (6), Bangladesh (5), Ethiopia (46), Peru (8) and Turkiye (9).Moreover, the Bovine Beadchip toolkit was used to evaluate the genomic diversity in the population of Colombian Chino Santandereano (14), Korean Jeju Black (2), German Black Pied (33) and Indonesian Bali cattle (39) cattle.Interestingly, many SNP markers in the Illumina 50K Bovine Beadchipwere used for genetic characterization in many cattle breeds (26).Commonly, three SNP markers ID of ARS-USMARC-Parent, ARS-BFGL-NGS, and BOVINEHD were recommended for the genetic characterization in cattle (22).Therefore, many SNP markers can discriminate between Bos indicus and Bos taurus cattle breeds (27).Furthermore, those SNP markers can be used for parentage checking in inbred cattle (13).Unfortunately, studies on Indonesian cattle breeds with SNP markers were not reported.Hence, this study aimed to characterize four Indonesian cattle breeds with Illumina Parentage SNP Markers.The results of this study are significant as the additional findings explain the kindship of Indonesian cattle breeds.

MATERIALS AND METHODS
Ethics approval: This study was approved by the Animal Ethics Committee of the Indonesian Ministry of Agriculture (Protocol No. Balitbangtan/Lolitsapi/Rm/11/2018).

Sample and DNA extraction
A total of one hundred and thirteen (113) mixed-sex cattle comprising Bali (16 heads), Madura (16 heads), Ongole grade (33 heads), and Sumba Ongole (48 heads) breeds were used for the experimental animals.The blood samples were taken from each animal from the jugular vein using a venoject vacutainer tube containing EDTA.The DNA extraction was performed using Genomic DNA Extraction Kit (Geneaid, Taiwan) following the manufacturer's instruction.Selecting SNP markers SNP Markers were obtained by using Illumina Bovine 50 K SNP BeadChip with the extracted DNA through a commercial laboratory service (Macrogen, South Korea).Therefore, two PLINK output files (pad and map) were obtained from the initial analysis with Genome Studio 2.0.software (48).A TASSEL 5.0.software (7) was also used to obtain the SNP markers from pad and map files.Subsequently, all the SNP markers belonging to SNP ID: ARS-USMARC-Parent (119 sites) and SNP ID: ARS-USMARC (37 sites) were selected for genetic characterization.In the present study, 156 sites of SNP markers were obtained from each cattle breed according to the Illumina Bovine 50 K SNP BeadChip.

Data analysis
The nucleotide data of 156 SNP markers were computed as the sequence data using BioEdit software (19).The sequence data changes to the numerical data are then used for discriminant analysis using SPSS 16.0 software with criteria of breeds as the factor and SNP markers as the variable.The discriminant analysis determined the individual discriminant plot based on the SNP markers and detected the discriminating SNP markers (38).At the same time, the UPGMA tree was performed using TASSEL 5.0 software for determining the kinship among Indonesian cattle breeds based on the Parentage SNP Markers.A MEGA software (20) was also used to calculate the pairwise genetic distance (F st ) in the animals under study.A structure software (10) (5).

Genetic characterization
The pairwise genetic distance (F st ) among four Indonesian cattle breeds is presented in Table 2.According to Table 2, Bali and Madura's cattle had a close distance (0.043/0.151) and similar to Ongole grade and Sumba Ongole (0.000/0.188).Generally, Madura has a lower genetic distance with Ongole lineage (0.032/0.251) than Bali (0.073/0.325).Lin et al. (27) reported a close distance among Myanmar, Laotian and Cambodian cattle (0.009 to 0.010) based on 58 SNP markers.These values are similar to the present study in Ongole grade -Sumba Ongole (0.000 to 0.010).In the African cattle breeds, the F st value with Illumina Bovine 50K BeadChip among Nguni, Drakensberg, and Bonsmarawas about 0.043 (30) and similar to Madura -Bali (0.043) in the present study.As a result, the discriminant plots of Ongole grade and Sumba Ongole cattle were located at a similar cluster, as shown in Figure 1.While, Bali and Madura cattle were located at the different cluster.Otherwise, the discriminant analysis revealed that twenty-three (23) SNP markers were detected as the discriminating SNP markers to characterize four Indonesian cattle breeds, as shown in Table 3.Additionally, the discriminant analysis with 156 SNP markers able to characterize four Indonesian native cattle with the canonical correlation of 95.20% (function 1) and 82.70% (function 2).Interestingly, the number of discriminating SNP markers with discriminant analysis was higher than that obtained by Zwane et al. (49) with Lewontin and Krakauer's (FLK) statistics test (23 SNP markers vs 11 SNP markers).Furthermore, the genetic admixture study revealed that Bali, Madura, and Ongole lineages had different genetic composition (Figure 2).According to Figure 2, Madura cattle had the genetic composition from Bali (Bos javanicus) and Ongole (Bos indicus) lineages (k=4).A similar finding was reported by Firdhausi et al. (12) that detecting two maternal lineages in Madura cattle, i.e., Bos javanicus and Bos indicus, based on mitochondrial Cyt-b and D-loop genes.Therefore, Ongole grade and Sumba Ongole cattle have two main genetic lineages based on the same parameters.Hartati et al. (16) reported that the Ongole grade cattle have two main genetic introgressions from Indian Zebu (Gir and Nellore) and Brahman cattle based on Illumina Bovine 50K BeadChip.Hence, two main genetic lineages in Ongole grade and Sumba Ongole may be originated from Indian Zebu and Brahman cattle.Agung et al. (1) supported it, which constructed the phylogenetic tree among Indonesian cattle breeds close to the present study based on 12 microsatellite loci.Additionally, the UPGMA tree revealed that Madura, Ongole lineage (Ongole grade and Sumba Ongole), Madura, and Bali cattle were characterized into their origin clusters, as shown in Figure 3.At the same time, the Ongole lineage cattle could be characterized into clade A (63 animals) and clade B (18 animals).Otherwise, the Ongole lineage cattle at clade B were close to the Madura cattle.Hence, the SNP markers under study can not discriminate between two different Ongole lineages clearly.However, Ongole grade and Sumba Ongole cattle can be discriminated against with their body measurements.Body length (BL), withers height (WH), hip height (HH), and chest depth (CD) in Ongole grade bulls were 123.97±11.58cm; 124.74±6.70 cm; 130.81±6.39cm and 58.63±5.94cm, respectively (21).These values were higher than BL, WH, HH, and CD in Sumba Ongole cattle, i.e., 112.50±10.12cm, 113.75±9.81cm; 120.50±6.35cm and 57.25±30.27cm, respectively (37).In the future, a depth study involving other Indonesian cattle breeds and many SNP markers will be essential to evaluate the potential genetics of Indonesian cattle breeds.

Table 1 . Genetic diversity in four Indonesian cattle breeds based on 156 sites of Parentage SNP Markers
was then used to observe the genetic admixture of animals under study with the numerical data.The genetic diversity in four Indonesian cattle breeds based on 156 sites of Parentage SNP markers was presented in Table1.The MAF value in Bali was lowest than other breeds.While Madura, Ongole grade, and Sumba Ongole cattle had a similar range of MAF value (0.14 -0.16).According to Illumina Bovine 50 K SNP Beadchip, the R (22.58%) was detected as the most frequent