THE SRY GENE VARIATIONS AMONGST SELECTED MADURA CATTLE POPULATIONS

The aim of this study was to determine the diversity of the sex-determining region Y (SRY) gene in Madura cattle bulls that had specifically selected for the production of frozen semen. DNA was isolated from the whole blood derived from 5 Madura cattle bulls. The SRY gene amplification was carried out using polymerase chain reaction (PCR) with SRY-4 as a primer followed by DNA sequencing and BLAST analysis to find out nucleotid variation and to construct filogenetic tree between Madura cattle bulls. Results revealed the proximity of the Madura cattle SRY genes to the Bos indicus SRY genes, furthermore this study also proved the existence of variation in Madura cattle SRY gene caused by mutation and deletion of nucleotides. It was concluded that the variations in Madura cattle SRY genes still persist even in the specifically selected populations with similar phenotype. ____________________________________________________________________________________________________________________


INTRODUCTION
Sex-determining region Y (SRY) gene has been known as testes genes and male sex determination factor.It was haplotypes and no recombination occurs during meiosis made it conserved and only inherited by the male lineage (Liu and Ponce de Leon, 2007).Moreover, the SRY gene diversity was reported low due to the slow rate of mutation (Mburu and Hanotte, 2005).Although genetic diversity of SRY gene was fairly low, SRY gene has a high enough diversity between species, has made it widely used to determine the origin of modern livestock from the sire lineage based on its proximity between species (Syed-Shabtar et al., 2013).
The diversity of genes, including the SRY gene would be exists in a natural population.Previous study has shown variations of SRY gene in one of Indonesian indigenous cattle species actually exist (Winaya et al., 2012).Similar research might be useful to observe the SRY gene variation in another indigenous cattle especially Madura breed that have been selected for frozen semen productions.Therefore, the aim of this research was to study the diversity of the SRY gene on the Madura cattle stud which has been selected for the frozen semen productions.

MATERIALS AND METHODS
The animals used in this study were 5 Madura cattle bulls that have been selected for frozen semen production in artificial insemination center in Singosari, Indonesia.The five bulls used in this study have passed the bull soundness examinations and have declared eligible as a stud and produce normally.

DNA Preparation
The DNA was isolated from the whole blood of five Madura cattle bulls by chloroform: Isoamyl-alcohol methods described by Sambrook et al. (1989).DNA amplification was carried out by PCR with primers of SRY-4: 5 '-GCC TGG ACT TTC TGC TTG TTA -3' and reverse 5 '-ACA GTG GGA GAC AAA ACA TAT -3' for Bos javanicus (Verkaar et al., 2003).All the stages from the DNA isolation to PCR was performed at Laboratory of Molecular Biology, University of Brawijaya while DNA sequencing was performed by Apical Scientific Sdn.Bhd.

Sequence Analysis
The NCBI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi#)sequence analysis was performed to compare sequences between bulls and species comparison by the alignment and build a phylogenetic tree.

RESULTS AND DISCUSSION
Y chromosome genes in male-specific region of the Y chromosome (MSY) did not recombine during meiosis, therefore the diversity within a species were relatively very small due to the low rate of mutations in Y chromosome genes, including SRY (Mburu and Hanotte, 2005;Nijman et al., 2008).Despite a low diversity within species, the SRY gene has a high diversity between species.For that reason, the SRY was widely used to determine an individual's genetic proximity into species level (Syed-Shabtar et al., 2013).
Madura cattle breeds was believed a croosbreed of two species of Bos indicus and Bos javanicus although phenotypically closer to the Bos indicus characterized by hump owned (Payne and Rollinson, 1976).Paternal lineage of Madura cattle was believed to have originated from Bos indicus (Nijman et al., 2003;Mohamad et al., 2009) while the maternal lineage originated from Bos javanicus (Kikkawa et al., 2003) although Verkaar et al. (2003) also reported that Bos javanicus Y chromosome has found in some Madura cattle bulls.
SRY gene was always being inherited through the paternal lineage; therefore, it can be used to trace the origins of a species from its proximity.The SRY-4 amplification in our study produced the 580 bp of Madura cattle bull SRY sequences.The BLAST phylogenetic analysis showed that the SRY gene of our Madura cattle bull were close to the Bos indicus SRY gene rather than Bos javanicus (Figure 1), despite of the primers used for DNA amplification derived from Bos javanicus SRY.On the subject of paternal lineage, these results were similar to those reported previously by Nijman et al. (2003) and Mohamad et al. (2009).This study indicated that the Madura cattle bull in our study has a paternal lineage derived from Bos indicus.
In spite of Madura bull SRY gene had a proximity to Bos indicus DQ 336527.2, the sequence alignment showed the difference in the base number 1815 ~ 1817; 2372; and 2389 between Madura bull and Bos indicus DQ336527.2.The diversity of SRY gene was found in the form of A-C mutations in 1815 bp; C-T at 2372 bp; and G-A at 2389 bp that known as a SNP.Beside the mutations, the deletion also found in 1816 ~ 1817 bp of SRY Madura cattle bulls compared to the reference SRY (Figure 2).
The diversity of the SRY gene can be occurred due to mutations, alterations and micro deletion (Mukherjee et al., 2013).The sequences analysis showed the deletion and mutation were exist in Madura bull SRY gene, indicating the diversity of SRY gene within Madura bulls (Figure 3).Based on our results, adenine deletion was found in 31 bp of bull number-30 while  SRY gene was known as testis forming genes that control the male sex differentiation (Li et al., 2014).The diversity of the SRY gene due to mutation and deletion were associated to infertility and genital developmental disorders symptoms (DSD syndrome) in the form of hermaphroditism (Lu et al., 2013).However, in this study the mutations and deletion could be found also in the SRY gene of stud with normal semen production.Nonetheless, it is very important to learn more about the diversity that found in the SRY gene of Madura cattle.

CONCLUSION
The proximity of the Madura cattle SRY gene to the Bos indicus SRY gene has shown the patrilineal relationship of zebu to Madura cattle.The specific selection for the frozen semen productions purposes was made the phenotypic uniformity amongst the bulls inside AI stations, nevertheless the diversity still occurred in the molecular levels that shown by the deletion and mutation in the SRY gene.Moreover, the diversity of SRY gene can lead the phylogenetic study to reveal the patrilineal ancestor of the modern Madura cattle.

Figure 3 .
Figure 3. Variation of SRY gene amongst Madura bull