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Proceedings of the World Congress on Genetics Applied to Livestock Production, 11.109

Detection of Single nucleotide polymorphism of Promoter SRY Gene on Bostaurus, Bos indicus and Bos sondaicus

T. Hartatik1,*, D. A. Priyadi1, Panjono2, Y. Adinata3

1Department of Animal Breeding and Reproduction, Faculty of Animal Science, UniversitasGadjah Mada, Yogyakarta, [email protected] (Corresponding Author)2Departemen of Animal Production, Faculty of Animal Science, Universitas Gadjah Mada.Jl. Fauna No. 3, Bulaksumur, Depok, Sleman 55281, Yogyakarta, Indonesia.Tel.: +62-274-4333373, Fax.: +62-274-5215783Beef Cattle Research , Grati, Pasuruan 6714, Indonesia

Summary

Improving the genetic quality of beef cattle in Indonesia has been done by introducingthe exotic breeds, in order to increase the production of national beef. The aim of thisresearch was to determine promoter sequence of SRY gene Bos taurus, Bos indicus and Bossondaicus. This study is necessary to control the seed population of local cattle. SRY genesare widely used to detect the paternal inheritance. Bos taurus cattle have a high percentage ofthe carcass and utilize feed with moderate quality efficiently. While Bos indicus cattle haveresistance to disease, extreme environments change, and utilize low-quality feed moreefficient. Bos sondaicus or Bali cattle is the specific germ plasm of Indonesian cattle whichtheir purity must be protected from the introduction of other breed. Twenty seven samplesconsist of ten Bos taurus, seven Bos indicus and ten Bos sondaicus were used in theseresearch. The target gene was a promoter region on SRY gene (1,281 bp). The SRY promotertarget sequence was obtain by polymerase chain reaction method using a specific primer(forward: 5'-GGA TTG ACA CAT TTG GCT GA-3 '; reverse: 5'-TTC TTA CCA CAG ACTGAC TTA GTGC-3') and then the PCR product was sequenced using both primer (twodirection sequences). The sequence results were aligned using ClustalW in BioEdit 7.1software to get the optimum size using two direct sequence result. The single pass sequenceusing the forward and reverse primer showed the size of 889 bp and 692 bp, respectively. Thecombination of both sequence alignment produce 1,175 bp. The restriction enzyme mappingof the sequence showed 14 restriction enzymes with 4-bases recognition site, 9 restrictionenzymes with 5-bases recognition site, and 15 restriction enzymes with 6-bases recognitionsite. Bos taurus shows the monomophic form of SRY promoter sequence. However, fivesingle nucleotide polymorphism was identified at position -966 C/G, -907 T/del, and -402C/T for Bos sondaicus, then at position -140 G/A and -117 G/A for Bos indicus. Singlenucleotide polymorphism at the promoter region of SRY gene in Bos sondaicus indicate thespecific marker, so it can be used to differentiate between Bos sondaicus and the other breeds,especially can be used to detect the crossbred cattle.

Keywords: Bos taurus, Bos indicus, Bos sondaicus, promoter, SRY gene, crossbred

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Introduction

Sex determination in mammals was determined by the presence or absence of the Ychromosome. The male sex appears by the normal functioning of the genes on Ychromosome, the sex-determining Y gene (Ruvinsky, 2015). Cow embryo with XYchromosome (male) shows faster gonadal differentiation, ie at 25-27 days of pregnancy,whereas females are formed a few days later (Juarez-Oropeza et al., 1995). The unexpressedof SRY will stimulate ovarian development (Ross et al., 2008). The section on SRY that hasbeen widely studied is the High mobility group (HMG) box, encoding a protein that is atranscription factor that can bind and bend DNA strands (Harley et al., 1992; Coriat et al.,1993). In-vitro testing shows that HMG boxes have the ability to bind to specific DNAsequences (AACAAT) and can bend DNA to 90° (King and Weiss 1993).

The 5 'and 3' prime UTRs are involved in regulating of SRY gene expression. The 5'UTR is composed of the main parts of which are the TATA boxes (Barrett et al., 2013),CAAT box, SRY-binding site, and Sp1-binding site (Cheng et al., 2001). The SNP on thepromoter, including the 5 'UTR and TATA box, there is evidence of differences intranscription activity between genotypes (Alam et al., 2012; Ham et al, 2010).

Polymorphism in SRY gene has been identified in Madura cattle (Hartatik et al.,2014), Friesian-Holstein, Sahiwal (Mukhopadhyay et al., 2011), Bali (Hassanin & Ropiquet,2007), and buffalo (Zhang et al., 2006). Polymorphism denotes a specific type of allele thatcan serve as an effective marker for detecting cross-breeding of cattle (Zhang et al., 2006),based on equality of mutations (polymorphism) inherited from parent to its offspring (Verkaaret al., 2004).

Material and methods

Animals

The animal used was 27 male cattle from three breeds, Bos taurus (n= 10), Bosindicus (n= 7) and Bos sondaicus (n= 10). These include two SRY genes (GenBank), fromHereford cattle (Bos taurus, Acc. No. AC232880.1) and Nelore cattle (Bos indicus No.NC_032680.1), which are also used to design the primers for PCR process.

Blood sample and DNA isolation

The samples of DNA were isolated from blood taken from the jugular or caudal veins.Blood was collected using a vacuum tube (Venoject) (DB, Indonesia) containing K3EDTA -anti-coagulant. The isolation of DNA was performed at the Genetics and BreedingLaboratory, Faculty of Animal Husbandry, Universitas Gadjah Mada using DNA Extractionkit (Genaid, Taiwan).

Polymerase chain reaction (PCR)

The primers were arranged based on two reference sequences (AC232880.1 andNC_032680.1) that were downloaded from GenBank databases (www.ncbi.nln.nih.gov). Apair of primers (forward 5'-GGA TTG ACA CAT TTG GCT GA-3, and reverse 5'-TTC TTACCA CAG ACT GAC TTA GTGC-'3) flank the target gene of 1,281 bp.


DNA sequencing and data analysis

The PCR product (30 μl/sample) and primer (5 μl/sample) sent to PT. GenetikaScience Indonesia to do sequencing. The sequences aligned using Bioedit software (version7.2.5) to identify the existence of polymorphism.

Results and discussion

Alignment of 27 sample promoter region SRY gene sequence showed the presence of5 SNPs. Three SNPs were revealed in the Bali cattle at nucleotide number -966 (C/G), -907(T/deletion), and -402 (C/T). Two SNP revealed in the Nelore cattle (Bos indicus; GenBankAcc. No. NC_032680.1) at nucleotide number -140 (G/A) and -117 (G/A). The other fifteensequences show a uniform result (Figure 1). Based on the SRY gene mapping (Figure 2), thepolymorphism does not placed in the parts that are known to be important in regulatingtranscription processes, such as the CAAT box, TATA box, SRY-binding and Sp1-bindingsections. This finding was similar to Cheng et al. (2001) results on various Bovidae families.

Figure 1. Alignment single-pass sequencing forward and reverse primer on SRY genepromoter region.


Figure 2. The SRY gene mapping

The upstream DNA region that influences the transcription process is CAAT box andthe TATA box. The CAAT box occasionally becomes the location of the polymerase enzymeto initiate the transcription and becomes the site to attach the transcription factors such asNF1 and SP1 (Kadonaga, 2004; Karp, 2010). The TATA box region has a more vital role, soit is referred to as one of the core promoters. The TATA box section is the location of apreinitiation-complex, consisting of RNA polymerase II and most transcription factorsrequired by the genes of eukaryotic organisms to be translated (Kadonaga, 2004; Karp, 2010;Kadonaga, 2012). The SRY-binding site is also a transcription activating sequence in thepromoter region (Matsuzawa-Watanabe et al., 2003), found in the rich A-T region (Harley etal., 1994). The other transcriptional regulatory of the promoter is a Sp1-binding site. That is atranscription factor exists in almost all genes. Serves as a basis in the continuity of thetranscription process, which mediates between the signal and the target genes that respond ina biological pathway (Samson & Wong, 2002). Single nucleotide polymorphism in thepromoter region is strongly possible to have consequences for the gene regulation (Li et al.,2013).

The determination of the males sex by SRY gene was initially believed to bedetermined solely by HMG-box activity, but further studies show that non-HMG-box regionalso plays a role in the expression of male sex (Zhao & Koopman, 2012). This is evidencedby the failure or disruption in the development of male gonads, resulting in sex-reversal ordecreased fertility by the presence of abnormalities in the promoter region of SRY gene(Tagliarini et al., 2005). Allegedly, abnormalities in the non-HMG-box region, resulting inthe disruption of target DNA binding affinities The unstable interaction with protein partnersis important in the transcription process (Zhao & Koopman, 2012). This is caused by thedisruption of SRY in performing its function as a transcription factor that triggering Sertolicell differentiation on testes (Chen et al., 2015). Although the actual interaction between therole of the promoter region as a transcription factor has not been fully revealed (Zhao et al.,2016).

The identical promoter region of SRY gene is also shown in different species, such asbetween goats and cattle (Cheng et al., 2001). The similarity is also seen in different speciesout of the families, indicating that the function of sex-determinant genes as a determinant of


the male sex is similar (Coriat et al., 1993). The similarity in the SRY gene reveals thehypothesis that it is possible that the gene is evidence of evolution, since it is known that sexchromosome may be derived from autosomes (Ellegren, 2011), and X chromosome firstlyformed, then differentiated into Y chromosome (Chang et al., 2001; Ellegren, 2011). It wassupported by the study that reveals the SRY gene is derived from the SRY-box gene family ofSOX3. The SOX gene can be found in both vertebrates and invertebrates (Cheng et al.,2001).

The Bos Genus is comprised of seven species: Bos taurus, Bos indicus, Bos sondaicus,Bos grunniens, Bos frontalis, Bos souveli (possibly extinct). In Indonesia, there are threespecies known exist, namely Bos taurus, Bos indicus and Bos sondaicus. Crosses betweenBos species is known to produce fertile offspring (Sutarno and Setyawan, 2016).Demonstrated with most of the Indonesian cattle with various phenotypes have mtDNA ofBos sondaicus. The Bos sondaicus is a native Indonesian cattle (Muhamad et al., 2009).

Restriction enzyme mapping indicated that there were 15 enzymes 4-base (Table 1), 9enzymes 5-base (Table 2) and 15 enzymes 6-base (Table 3) that could cut the SRY genes.However, only BfaI restriction enzyme can recognize the polymorphism in SRY gene.Precisely at SNP-966 (C/G) that identified in Bali cattle. The enzyme can be used to identifythe SNP in further research.Table 1. Restriction enzyme with 4-base recognition siteNo Enzyme Recognition site Frequncy Position1 AluI AG’CT 4 410, 775, 804, 11112 BfaI* C’TA_G 1 9553 BstKTI G_AT’C 2 49, 4444 Csp6I G’TA_C 2 630, 7885 DpnI GA’TC 2 48, 4436 FatI ‘CATG_ 2 245, 8467 HpyCH4IV A’CG_T 2 786, 11598 HpyCH4V TG’CA 6 11, 44, 463, 491, 807, 9699 MboI ‘GATC_ 2 46, 44110 MseI T’TA_A 10 28, 207, 608, 655, 671, 705, 91311 NlaIII _CATG’ 2 249, 85012 RsaI GT’AC 2 631, 78913 TaiI _ACGT’ 2 789, 116214 Tsp509I ‘AATT_ 10 77, 94, 338, 372, 385, 393, 67715 MaeI C’TA_G 2 403, 999

Table 2. Restriction enzyme with 5-base recognition siteNo Enzyme Recognition site Frequncy Position1 BstNI CC'w_GG 2 615, 11552 Fnu4HI GC'n_GC 2 805, 9673 Hpy188I TC_n'GA 5 197, 567, 693, 735, 10604 HpyCH4III AC_n'GT 4 177, 351, 563, 9425 MaeIII 'GTnAC_ 2 180, 3806 PspGI 'CCwGG 2 613, 11537 ScrFI CC'n_GG 2 615, 11558 StyD4I 'CCnGG 2 613, 11539 TseI G'CwG_C 2 804, 966w= A/T; n= G/C/A/T


Table 3. Restriction enzyme with 6-base recognition siteNo Enzyme Recognition site Frequncy Position1 AccI GT'mk_AC 1 10252 Acc65I G'GTAC_C 1 6293 ApoI r'AATT_y 3 94, 393, 10544 BanI G'GyrC_C 1 6295 BclI T'GATC_A 1 4416 BsaAI yAC'GTr 1 7877 BsaJI C'CnnG_G 2 614, 6338 DraI TTT'AAA 1 2089 HindIII A'AGCT_T 1 40810 Hpy8I GTn'nAC 3 264, 945, 107111 KpnI G_GTAC'C 1 63312 MfeI C'AATT_G 1 37213 PsiI TTA'TAA 1 40514 PvuII CAG'CTG 1 80415 SnaBI TAC'GTA 1 787m= A/C; k= G/T; y= C/T; r= G/A; n= G/C/A/T

The SNPs revealed in the promoter region of Bali cattle SRY gene can be used toevaluate the purity of the cattle that are traced from the paternal path. It can be used tosupport the breeding program of Bali's pure cattle which is the Indonesia native cattle.

Conclusion

Five SNPs were found in the promoter region of cattle SRY gene. Three SNPsrevealed on Bali cattle (-966 (C/G), -907 (T/deletion), -402 (C/T)), and two SNPs revealed onNelore cattle (-140 (G/A) and -117 (G/A)). Polymorphisms in Bali cattle (SNP-966 C/G) canbe recognized by BfaI restriction enzymes.

Acknowledgment

This research was the result of a cooperation between Faculty of Animal ScienceUniversitas Gadjah Mada with PT. Widodo Makmur Perkasa, Cileungsi, Bogor, Indonesia.Partially funded by a Competence Based Research Grant (No. Kontrak KP2296/UN1.P.III/DIT-LIT/LT/2017). Thank you to Riyan Nugroho Aji and Retno Setyawatifor helping collect the blood samples and for helping the laboratory analysis.

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