SRY Gene Marker Differences in Native and Crossbreed Cattle

1Department of Animal Breeding and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia 2Technology of Processing Livestock Product, Politeknik Negeri Banyuwangi. Jawa Timur, 68461, Indonesia 3Departemen of Animal Nutrition and Feed Science, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia 4Departemen of Animal Production, Faculty of Animal Science, Universitas Gadjah Mada. Yogyakarta, 55281, Indonesia 5Beef Cattle Research Station, Grati, Pasuruan, 6714, Indonesia


Introduction
The existence of Y chromosomes determines typically the sex in mammals.Male sex would be determined by the normal function of the Y chromosome gene or Sex-determining region Y (Ruvinsky, 2015).Sex chromosomes (X and Y on mammals) are widely discussed as proof of genetic evolution (Ellegren, 2011).It could be seen from the similarity on the promoter of the SRY gene from different animal species, like in goat and cattle (Cheng et al., 2001).The similarity could also be seen from species in the different family, thus indicates a similar sex-determining gene function for the male sex determinant (Coriat et al., 1993).The sex chromosome could be originated from the autosome (Ellegren, 2011), with the X chromosome formed earlier then differentiated into Y chromosome (Chang et al., 2001;Ellegren, 2011).The theory is also supported by the finding of the SRY gene from SRY-box family, which is the SOX3.Furthermore, the SOX gene is currently found whether in vertebrate or invertebrate (Cheng et al., 2001).
A cattle embryo with XY chromosome (male) would show a faster gonad differentiation, at 25 -27 days of pregnancy, while for a female the differentiation occurred several days later (Juarez-Oropeza et al., 1995).The unexpressed SRY gene then stimulates the ovary development (Ross et al., 2008).Most researches have been done on the High mobility group (HMG) box of SRY, a protein which acts as a transcription factor that binds and bends the DNA strands (Harley et al., 1992;Coriat et al., 1993).From the in vitro analysis, it can be seen that the HMG box has the ability to bind specific DNA sequence (AACAAT) and could bend the DNA up to 90 o (King dan Weiss, 1993).
The 5'-end and 3'-end of UTR are involved in the SRY gene expression.The TATA box is the main part which composed the 5'-end of UTR (Barrett et al., 2013).Cheng et al. (2001) added that the most important part of 5' UTR regarding the SRY gene regulation are CAAT box, TATA box, SRY-binding site, and Sp1-binding site.Research by Alam et al. (2012) and Han et al. (2010) showed that the existence of SNP on the promoter region which includes 5' UTR and TATA box indicates a different transcription activity between the genotypes formed by the SNPs.
SRY gene polymorphism on the coding region has already been identified on Madura cattle (Hartatik et al., 2014), Friesian Holstein cattle, Sahiwal cattle (Mukhopadhyay et al., 2011), Bali cattle (Hassanin dan Ropiquet, 2007), and buffalo (Zhang et al., 2006).The polymorphism showed specific alleles type which can be used as an effective genetic marker to detect crossbreeding between livestock (Zhang et al., 2006), based on the inherited mutation (polymorphism) similarity (Verkaar et al., 2004).The promoter region of the SRY gene on native cattle or its crossbreeds has never been studied before.Thus this study becomes important to further understand the promoter region of the SRY gene of the breeds.

Blood samples and DNA isolation
DNA samples were isolated from blood, which were taken through the jugular or caudal vein.The blood samples were collected using a vacuum tube (Venoject, DB Indonesia) which contained K3EDTA anti-coagulant.DNA isolation was done in the Laboratory of Animal Breeding and Genetics, Faculty of Animal Science, Universitas Gadjah Mada by using DNA Extraction kit (Genaid, Taiwan).

DNA amplification (PCR)
Primers (Table 1) were aligned based on two reference sequences (Acc.No. AC232880.1 dan NC_032680.1)downloaded from Genbank database (www.ncbi.nln.nih.gov).Primer pairs (forward 5'-GGA TTG ACA CAT TTG GCT GA-'3, and reverse 5'-TTC TTA CCA CAG ACT GAC TTA GTGC-'3) flank the target gene at 1,281 bp (Figure 1).DNA sequencing and data analysis PCR products as much as 30 µl/sample and 5 µl/sample of primers were sent to PT. Genetika Science Indonesia for sequencing.The obtained sequences were then aligned by using Bioedit software (7.2.5 version) to identify the existence of polymorphisms.A phylogenetic test was done based on the SRY gene sequence (1,281 bp) by using Mega 5 software, and the construction was done by using Maximum likelihood tree (1000 Bootstrap repetitions).

Result and Discussion
Five SNPs were obtained from the promoter region of SRY gene sequences alignment of 19 cattle.Three SNPs revealed on Bali cattle were at -966 C/G, -907 T/deletion, and -402 C/T, located before CAAT box, TATA box, SRY-binding, and Sp1-binding region.Two SNPs revealed on Nellore cattle (Bos indicus; GenBank Acc.No. NC_032680.1)was at -140 G/A and -117 G/A (Table 2), located after CAAT box, TATA box, SRY-binding, and Sp1-binding region.
Fifteen other sequences showed similar results on its nucleotide compilers.Polymorphism was not located on the important region for transcription processes such as CAAT box, TATA box, SRY-binding, and Sp1-binding.This finding is in accordance with Cheng et al. (2001) who observed a different family of Bovidae and found the SNPs on the promoter region that did not regulate the transcription process.However, single nucleotide polymorphisms which were found outside the transcription regulators might have certain consequences to the genetic regulation levels, regarding that the function between promoters is interrelated (Li et al., 2013).The upstream DNA region which was already known to affect transcription process is CAAT box, as it is the location for polymerase enzyme to initiate the transcription process, while also acts as the binding site for transcription factors such as NF1 and SP 1 (Kadonaga, 2004;Karp, 2010).The TATA box region plays a more vital role so that it is considered as the core promoter.The TATA box region is the location of the preinitiation complex which consisted of RNA polymerase II, while also acts as the binding site for most transcription factors required for the gene in eucaryotes to transcript DNA sequences (Kadonaga, 2004;Karp, 2010;Kadonaga, 2012).The SRY-binding site also acts as sequence transcription activators on the promoter region (Matsuzawa-Watanabe et al., 2003), while some parts are rich of A-T base nucleotides (Harley et al., 1992).The other transcription regulator on the promoter region is the Sp1-binding site, which exists in almost all genes.The Sp1-binding site acts as the base for the transcription process, mediating between signal and target genes which responded in one biological stream process (Samson dan Wong, 2002).Five SNPs found on the promoter region of SRY gene could impact the gene expression normality as each was interrelated (Li et al., 2013), and the occurred interaction on each part might impact the overall gene expression.
The existence of SNP in the promoter region of the SRY gene has been known to affect the genetic expression of the male sex.It is initially believed that the expression would be only determined by the HMG-box activity.However, further researches showed that the non-HMG-box also plays a certain role on the male sex expression (Zhao and Koopman, 2012).It can be seen from the abnormality in the promoter region of SRY gene which could cause failure or disturbance on the male gonad development, thus causing sex-reversal or lower fertility (Taqliarini et al., 2005).It is strongly suggested that the abnormality on the non-HMG-box would disturb the DNA target binding affinity and caused unstable interaction between the proteins responsible for transcription (Zhao and Koopman, 2012).On the other hand, the disturbance could also cause the disturbance in the SRY gene, a transcription factor, which initiate the Sertoli cell differentiation on testicle (Chen et al., 2015), even though the exact interaction between promoter region as a transcription factor is yet to be found (Zhao et al., 2016).
Bos genus consists of six species, namely Bos taurus, Bos indicus, Bos sondaicus/Bos javanicus, Bos grunniens, Bos frontalis, Bos souveli (possibly extinct) (Wilson et al., 2005).In Indonesia, there are three Bos species exist, which are Bos taurus, Bos indicus, and Bos sondaicus.A crossbreeding between those Bos species is known to yield fertile offspring (Sutarno dan Setyawan, 2016), which can be seen from the most cattle in with various phenotypes which have Bos sondaicus mtDNA is the Indonesia native cattle (Mohamad et al., 2009;Priyadi et al., 2017).
Phylogenetic test on 19 cattle was divided into four groups; Bos indicus (Nellore; Acc.No. NC_032680.1),Bos taurus (Hereford; Acc.No. AC232880.1),14 samples (homogenous samples), and Bos sondaicus.Ovis aries (AF026566.1)was used as an outgroup species in reconstructing the phylogenetic tree.The result of the phylogenetic tree reconstruction (Figure 2) showed that Bos sondaicus reside on different branch compared to the other three groups.
Cattles which were included in the 14 samples group showed a homogeneity on the promoter region of the SRY gene (1,281 bp).It can be said that the bulls of the group are Bos taurus, even though the occurred phenotype is currently tended to resemble Bos indicus (humped), in this case, were Madura and PO cattle.The similar situation also is shown in  The study to determine SNP on the promoter region of Bali cattle SRY gene could be widely applied to check the cattle purity in Indonesia by tracing from the paternal line.Furthermore, it can also be used to support the breed purification program for Bali cattle as the Indonesia native cattle.Further research should be done with wider analysis area of the SRY gene, while also adding the research samples so that more comprehensive results can be obtained.

Conclusions
Five SNPs were found in the promoter region of the SRY gene.Bali cattle showed three SNPs (-966 C/G, -907 T/deletion, -402 C/T), while two SNPs were found in Nellore cattle (-140 G/A dan -117 G/A).Wagyu-BX, BX, Simmental, Limousin, PO, Madura, and Hereford cattle showed a uniform promoter region of the SRY gene.

Figure 2 .
Figure 2. Phylogenetic tree based on the promoter region of the SRY gene (1,281 bp).

Table 2 .
Nucleotide changes on the promoter region of SRY gene