Genetic characterization and lineage analysis of Pasundan (Rancah) cattle in West Java, Indonesia: Insights from ZFY gene sequencing

Objective: This study was carried out to characterize Pasundan (Rancah) cattle from West Java of Indonesia based on a partial Zinc Finger Y-linked (ZFY) gene sequence. Methods: Total of fourteen (14) DNA samples of unrelated Pasundan bulls at the breeding station (BPPIBT-SP Ciamis, West Java) were used for sequencing analysis. The blood samples were collected from jugular vein of each bull for the DNA extraction analysis. Along 979 bp of ZFY gene was amplified using primer pairs of forward: 5’ - GGT GAG GGC ACA TGA GTT C - 3’and reverse: 5’ - CTC TGC AGG TGG TTG TGT AA - 3’. Therefore, a forward sequencing method was performed in the present study to obtain the ZFY gene sequences. Along 714 bp of bovine ZFY gene sequences were used for determining the Neighbor-Joining (NJ) tree for Pasundan bulls with 1000 × bootstrap replications using a MEGA computer program. The genetic diversity parameters of haplotype diversity (H d ), nucleotide diversity (p i ), Tajima’s D test and Fu’s Fs statistics were calculated using a DNAsp computer program. Results: Total of fourteen (14) haplotypes of ZFY gene and seventy (70) mutation sites were detected in the Pasundan bulls. The haplotype diversity (H d ) and nucleotide diversity (p i ) in Pasundan’s ZFY gene diversity were 1.00 (high) and 0.01 (low), respectively. The neutrality test of Tajima’s D and Fu’s Fs statistic values were -1.42 and -6.47, respectively. Bovine ZFY gene can be classified into two clades of ZFY-1 and ZFY-2. Therefore, the Neighbor-joining (NJ) tree (1000×bootstrap) revealed that the Pasundan cattle are classified into clade ZFY-1 together with Bos taurus (AF241273, AF465179). Meanwhile, a clade ZFY-2 consisted of Bos taurus (AF465181, DQ336536), Bos indicus (DQ336537), and Bos javanicus (DQ336578). Conclusion: The ZFY gene of Pasundan bulls were polymorphic with seventy mutation sites. Therefore, Pasundan bulls were characterized into ZFY-1 clade or Taurine lineage based on ZFY gene sequence variaton. The crossbreeding program with Bos taurus straw to increase meat production of cattle may affecting the genetic structure of Pasundan bulls since this program is widely applied in West Java through many years ago.


INTRODUCTION
Pasundan (Rancah) cattle is one of Indonesian native cattle that originated from West Java Province of Indonesia.Pasundan cattle have been decided as the Indonesian native cattle through the decision of the Indonesian Ministry of Agriculture No: 1051/Kpts/RI/SR.10/2014[1].Generally, Pasundan cattle are kept as beef cattle with an adult weight of 309.50±57.37kg for males and 148.15±14.45kg for females [2].Pasundan bulls are able to reach 216.00±26.3kg of slaughter weight; 110.75±16.5 kg of carcass weight and 53.42±2.08% of carcass percentage [3].According to the mitochondrial DNA (mtDNA) of Cytochrome-b (Cyt-b) and Dloop genes, Pasundan cattle were closed to Bos javanicus [4,5].In addition, Pasundan cattle was closed to Madura cattle (Bos indicus) based on the mitochondrial COI gene [6].Unfortunately, a study to characterize Pasundan cattle based on Y-chromosomal diversity is not reported before.The Zinc Finger Y-linked (ZFY) gene is one of Ychromosomal genes that used for characterization in Bovidae family species [7,8].In the cattle, ZFY gene has the length of 20,780 bp with seven (7) exonic regions (GenBank: AF465181).Despite detecting paternal lineage, the ZFY gene can be used to identify the typical sex of buffalo [9].In addition, the ZFY gene can be used to detect the presence of Y chromosome in the cow that affecting infertility [10].
Genetic characterization of livestock animals is important to describe their genetic composition.Therefore, genetic diversity is usefull to characterize the breeds of livestock based on molecular information.Moreover, Ychromosome diversity is important to elucidate patterns, timing of speciation and phylogeography and hybridization events [7].
Previous studies have been determined the paternal origin of Lakor goat [11], Nigerian cattle [12], Bharal sheep [13] and Cinta Senese pig [14] based on ZFY gene sequence.This study aimed to characterize Pasundan cattle based on partial ZFY gene sequence.Despite this, the results in the present study had the potency for determining the genetic marker of reproductive traits in Pasundan bulls in the future.

DNA samples
A total fourteen (14) blood samples from different adult Pasundan bulls were collected for the analysis.All Pasundan bulls in this study were kept at the breeding station (BPPIBT-SP Ciamis) in West Java, Indonesia.The blood samples (about 5 mL) were taken from jugular vein using venoject and vacutainer tube containing EDTA.The DNA extraction was assessed with Genomic DNA Extraction Kit (Geneaid, Taiwan) following the manufacturer's protocol.

Amplification of ZFY gene and sequencing
Amplification of ZFY gene was performed in a total volume of 30 μL containing 9 μL of DNA template; 0.6 μL of each primer; 15 μL of PCR mix (Mytaq HS Red Mix, Bioline, UK) and 4.8 water nuclease free.Thermocycling conditions for ZFY gene were as follows: pre-denaturation at 95°C for 5 minutes, followed by 35 amplification cycles of denaturation at 95°C for 30 seconds; annealing at 55.4°C at 1 minutes 30 seconds; extension at 72°C at 30 seconds and final extension at 72°C at 5 minutes.Therefore, the PCR products (5 μL) were electrophoresed through 1% agarose gels (1 g of agarose powder and 100 mL of 1 x Tris Buffer EDTA) and stained with SYBR TM Green (1 μL per 10 was used in this study to amplify bovine ZFY gene (GenBank: AF465181) along 979 bp.Thus, the primer pairs in this study are able to amplify the ZFY gene from intron 6 to exon 7 regions (Figure 1).In addition, the forward sequencing analysis was performed in the present study by the 1 st Base Laboratory Service of Malaysia.

Sequence analysis
The sequence analysis was performed with three (3) molecular softwares of BioEdit [16], MEGA11 [17] and DNAsp [18].BioEdit software was used for sequence alignment and contig analysis.MEGA-X software was used to obtain the phylogenetic tree using Neighborjoining (NJ) method (1000×bootstrap) with the sequence references of Bos taurus (AF241273, AF465179, AF465181, DQ556336), Bos indicus (DQ336537) and Bos javanicus (DQ336538).A DNAsp software was used to obtain number of mutation site, Haplotype diversity (Hd), nucleotide diversity (pi), neutrality test of Tajima's D and Fu's Fs statistics tests.

RESULTS
Along 979 bp of ZFY gene amplicons was successfully amplified in the 1% of agarose gel as shown in Figure 2.However, 778 bp of partial ZFY gene sequence of Pasundan bulls is able to evaluate based on the chromatogram graphics.In addition, total 48 mutation sites (excluding indel mutation) were detected in the 714 bp of Pasundan ZFY gene as shown in Figure 3.A total of seventeen ( 17) mutation sites were detected in the exon 7 region of bovine ZFY gene.Therefore, total 52 amino acids were detected in the partial exon 7 region of bovine ZFY gene with 12 amino acid changes (23%) as shown in Figure 4. Hence, these mutation sites can classify the bovine ZFY gene into ZFY-1 and ZFY-2 clades.Moreover, the Neighbor-joining tree revealed that Pasundan cattle are classified into ZFY-1 cluster together with Bos taurus (AF241273, AF465179) as shown in Figure 5. Therefore, Bos javanicus ,Bos indicus and the other Bos taurus (DQ336536, AF465181) are classified into ZFY-2 cluster.The genetic diversity parameters of partial ZFY gene in Pasundan bulls were presented in Table 1.Therefore, a total of fourteen ( 14) haplotypes were detected in the animals under study based on the mutation sites as shown in Figure 3.The Hd and pi values in this study were 1.00 (high) and 0.01 (low), respectively.Thus, the neutrality tests of Tajima's D and Fu's Fs statistics were -1.42 and -6.47, respectively.

DISCUSSION
The genetic diversity in ZFY gene of Pasundan cattle are high with 48 polymorphic sites and Hd value of 1.00.Nei and Kumar [19] stated that Hd is classified into low (<0.50) and high (>0.50)categories.The Hd value can be influenced by outbreeding with the same breeds from different geographical regions.Currently, the government of West Java was distributed frozen sperm (straws) from many Pasundan bulls for purebreeding program in West Java province with artificial insemination (AI).Consequently, the Hd value in Pasundan bulls can be increased because of straws distribution.Despite this, hybridization between different sub-species also influences the genetic diversity of species [7].Actually, Pasundan is an indigenous cattle with two genetic introgresions of Bos indicus and Bos javanicus based on mitochondrial Dloop [5] and COI [20] genes.Nonetheless, The pi value in this study was 0.02 and classified into the low category.The pi value can be classified into low (0.01 -0.04), medium (0.05 -0.07) and high (0.08 -0.10) categories [19].In this study, the neutrality test (Tajima's D and Fu's) showed in a negative value and indicating that low nucleotide variation in Pasundan cattle was caused by species expansion [19].Arifin et al. [21] reported that the migration of Pasundan cattle from Majalengka to Sumedang and Indramayu was increased the allelic frequency of bloods albumin protein.Contrastly, Chaichanatong et al. [8] found a low genetic diversity in ZFY gene of Banteng (Bos javanicus) at two different geographical regions of Thailand.In addition, Clawson et al. [15] found a polymorphic site in ZFY gene of US beef cattle (Bos taurus) i.e. g.610C>T (GenBank: AF465181) but its was not found in Pasundan cattle (data not shown).

Figure 4 .Figure 5 .
Figure 4. Amino acid changes in the partial exon 7 gene of bovine ZFY gene region [27]  reported that Pasundan and Madura (Bos indicus) cattle have a similar genetic admixture based on twelve (12) microsatellite markers.CONCLUSIONThere are two (2) clades of bovine ZFY gene i.e.ZFY-1 and ZFY-2 based on partial exon 7 diversity.All Pasundan bulls under study were classified into ZFY-1 cluster together with Bos taurus.Hence, the exon 7 region of ZFY gene can be used as the genetic marker to discriminate Pasundan cattle with Bos javanicus and Bos indicus cattle breeds.

Table 1 .
The