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Procedia Environmental Sciences 20 (2014) 365 – 369

4th International Conference on Sustainable Future for Human Security, SustaiN 2013

Analysis of DNA Polymorphism in SRY Gene of Madura Cattle
Populations
Tety Hartatika*, T.S.M. Widib, S.D. Volkandaria, D. Maharania, Sumadia
a
Laboratory of Animal Breeding, Faculty of Animal Science, UGM, Yogyakarta, 55281, Indonesia
b
Laboratory of Draught and Companion Animal, Faculty of Animal Science, UGM, Yogyakarta, 55281, Indonesia

Abstract

The aim of this research was to assess the SRY-gene polymorphism in Madura cattle populations and the hybrids of Madura cow
with Limousin bull. We investigated the specific markers of the paternally transmitted Y-chromosome for the direct detection of
male-mediated introgression. We performed polymerase chain reaction and restriction fragment length polymorphisme (PCR–
RFLP) to identify the polymorphism of Y-chromosomes in 17 Madura cattle from Pamekasan district, 4 conserved Madura cattle
from Sapudi Island and 22 Limousin x Madura crossbred cattle from Pamekasan. A 211 bp PCR product were cut with PstI and
BfaI restriction enzyme. The result showed all the Madura cattle types were identified by PstI resrtiction enzyme performed into
two fragments DNA. The polymorphisme of Y-chromosome was recognized by BfaI restriction enzymes at the position 432 bp
and 544 bp. Using BfaI restriction enzyme, this study identified 68.18% of Limousin x Madura crossbred cattle was in the
sequence of Y-chromosome. The high numbers of BfaI site restriction in the sequence of Y-chromosome due to the introduction
Limousin bull by artificial insemination. For those, the preservation of pure Madura cattle as one of animal genetics resources in
Indonesia should be a concern.

© 2013
© 2014 The
The Authors.
Authors. Published
Published by
by Elsevier B.V. Open access under CC BY-NC-ND license.
ElsevierB.V.
Selection and
Selection and peer-review
peer-review under
under responsibility
responsibility of
of the
the SustaiN
SustaiN conference
conferencecommittee
committeeand
andsupported
supportedby
byKyoto
KyotoUniversity;
University;(RISH),
(RISH), (OPIR), (GCOE-ARS) and (GSS) as co-hosts
(OPIR), (GCOE-ARS) and (GSS) as co-hosts.

Keywords: Polymorphism; SRY Gene; Madura Cattle; PCR-RFLP

* Corresponding author. Tel.: +62-274-4333373; fax: +62-274-521578.
E-mail address: tetyharuta@yahoo.com.

1878-0296 © 2014 The Authors. Published by Elsevier B.V. Open access under CC BY-NC-ND license.
Selection and peer-review under responsibility of the SustaiN conference committee and supported by Kyoto University; (RISH), (OPIR),
(GCOE-ARS) and (GSS) as co-hosts
doi:10.1016/j.proenv.2014.03.046

which have previously lived in Java Island before migrating to Madura Island [1. 2. PCR products were first separated by electrophoresis on a 1% agarose gel. then 5 μl of the PCR product was digested by the addition of 5–10U of either PstI or BfaI restriction enzyme (New England Biolabs. respectively [5]. Since 2001. Universitas Gadjah Mada. However. Y-Chromosome is useful for analyzing the paternal lineages of the crossbred cattle population (e. It may have an impact on the formation of local breeds and could also affect the genetic integrity of them.1. crossbreeding with Limousin has been allowed. For detection of the Madura hybrid cattle Y-chromosome. MA. Limousin x Madura). Then. The introgression leads to increased genetic variation in the population. it can increase the conservation of animal genetic resources in Indonesia. Bos indicus. banteng and Madura cattle. The results of this research may be able to provide a basic molecular data to the germ plasm in identification of the characteristic of Madura cattle.366 Tety Hartatik et al.2. The program consisted of an initial denaturation for 5 min at 94°C followed by 35 cycles of 1 m at 92°C. 4 conserved Madura cattle from Sapudi Island and 22 Limousin x Madura (crossbred cattle) were used in this research. Sumenep Regency.25U Taq DNA polymerase (Promega. Limousin was preferred over Madura by Maduranesse due to its red colour. WI. Sample collection and DNA extraction Three types of cattle consisted of 17 Madura cattle from Pamekasan District. The cytochrom c oxidase I (COI) mitocondrial gene and SRY of Y chromosome were used to portray the phylogenetic relationships among five species of the Bos genus (B. The previous study [2] has analyzed DNA from zebu. Faculty of Animal Science. DNA was isolated from their blood samples by using standard SDS/proteinase K extraction [7]. Material and Methods 2. 1 m at 60°C and 1 m at 72°C and by a final extension for 7 min at 72°C. In restricted area of Madura. Bos gaurus. The analysis of mitochondrial. Introduction Madura cattle are one of the indigenous breeds maintained in Madura Island of Indonesia. Bos taurus. Because of the local climate adaptation and geographical condition.5 mM MgCl2. The samples were digested for 3 h at 37°C and . javanicus.g. They found Madura cattle to have characteristics of Zebu and Banteng. The use of artificial insemination using imported breed leads to the loss of indigenous breeds and its local environment adaptation. and protect uncontrolled crossbreeding in conservation area of Madura cattle (Sapudi Island). the Javanese cattle in Madura Island were isolated and become Madura cattle. Madura cattle are the same as Javanese cattle.2]. the hybrid of Limousin and Madura became more popular among farmers and the farmers in this area tend to cross Madura cattle with Limousin during the last 10 years. Y- chromosomal DNA markers are expected to have another geographical distribution and paternally inherited markers would reveal male introgression via breeding management with natural matting and artificial insemination as well. the preservation of Madura cattle as national genetic resources should be a concern.2 mM dNTP and 1. Though it is useful for upgrading of the breed. 2. Isolation and analysis DNA samples were conducted at Laboratory of Animal Breeding. USA) and the reaction buffer of manufacture’s recommendation. The analysis of mtDNA recognized that Madura cattle and Galekan Zebu indicated having 56% and 94% of Banteng. AB039748 for bovine SRY [8]. The investigation using molecular approach to indentify the native cattle had been widely observed. This research investigated Y-chromosomal DNA markers for several types of Madura cattle (exotic crossbreds and purebred) using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Beverley. Moreover. 0. Madura cattle had been recognized as crossbred between a Bos taurus or Bos javanicus cow and a Bos indicus bull [3]. The uniformity of Madura cattle was created through a continuous selection by the people at Madura [4]. Madison. and Bos grunniens) [6]. Y-chromosomal and microsatellite DNA had been used to describe the genetic architecture of Indonesian cattle including Bali and Madura [5]. PCR-RFLP The numbering of primer binding sites and restriction sites referred to the Genbank No. USA). / Procedia Environmental Sciences 20 (2014) 365 – 369 1. we used 50 ng genomic DNA in a total volume of 25 μl and performed the PCR using one of two pairs of primers: SRY-F2 (5’-GTT GAT GGG TTT GGG CTG ACT) and SRY-R2 (5’-AAA TTG AGA TAA AGA GCG CCT) with 1.

which produce 2 band and 3 band (Table 1). There were 68. Previous studies described the sequence variation in the Y-chromosomal SRY gene for the design of convenient tests of paternal lineages in bovine populations. / Procedia Environmental Sciences 20 (2014) 365 – 369 367 were electrophoresed on a 10% polyacrylamide gel to analyze restriction-fragments. Tety Hartatik et al. we recognized PstI restriction enzymes at the position 623 bp and BfaI restriction enzymes at the position 432 bp and 544 bp (Fig. The result of this study shows that both of Madura cattle in Pamekasan district and Sapudi Island have one pattern of restriction site. The occurrence of different type of Y chromosome on crossbred cattle showed the contribution of Bos taurus gene through the paternal line. Fig. Results and Discussion The product size of SRY gene was 928 bp (Fig. 4). while BfaI has two restriction types. Based on the sequence result by Macrogen Company. 1 The 928 bp PCR product size of SRY gene (GenBank Acc. Mapping of restriction enzyme (RE) on Bos taurus sequence showed that RE PstI has one restriction site while BfaI has two restriction site which produce 2 band and 3 band. . respectively. since it is the only single-copy gene without a X-chromosomal homologue [9]. AB039748) and the location of restriction site of PstI enzyme and BfaI enzyme. we performed DNA sequencing. SRY is the most obvious target for these assays. To confirm the PCR product. 1) and the result of DNA amplification by PCR method illustrated on Figure 2. 3.No. 3). which produces 582 bp and 346 bp (Fig. The number showed that the consistent using of Limousin bull can continually shift the genetic trait from Y chromosome on local cattle. There is one PstI restriction type. The different result was showed on crossbred cattle. The restriction mapping of BfaI and PstI site was constructed by BioEdit.18% change of sequence type of Y Chromosome in crossbred cattle.

Part of SRY sequence (a). 3 PCR-RFLP product of SRY2 gene using PstI restriction enzyme in Madura Cattle (M=Marker. 6-7= Bali cattle. Fig. Line 1-4 = PCR product) Fig. 4 Restriction mapping of SRY gen in Limousin x Madura cattle using BioEdit. 8-9=Madura cattle from Pamekasan.368 Tety Hartatik et al. b. chromatogram (b) and the position of BfaI dan PstI site restriction. 2 PCR product of SRY2 gene (M= marker. Product of PstI digestion in line 2-5= Madura cattle from Sapudi Island. / Procedia Environmental Sciences 20 (2014) 365 – 369 Fig. 1=PCR product. . 10=crossbred cattle) a.

Barwegen MW. Conclusion The introduction of Limousin bull by artificial insemination has improved the numbers of BfaI site restriction in the Y-chromosome sequence. 4 (100%) (100%) Limousin x Madura Cattle Pamekasan 22 22 15 7 (100%) (68. Rosli MK. Hanotte O. USA. 47 (1): 2-6. Jakarta. 8 Kato Y. Tety Hartatik et al. Yaakop S. Olsson M. 3 Popescu CP and Smith WG. all the Madura cattle type were identified by PstI resrtiction enzyme performed into two fragments DNA (two DNA band). but was also supposed as a result of zebu–banteng hybridization [10]. the BfaI restriction enzyme had digested into three fragments (Table 1). 4 Directorate General of Livestock Service. Lenstra JA. Animal Breeding for Cattle. Cold Spring Harbor Laboratory Press: Cold Spring Harbor. Sutou S.Bio. Colenbrabder B. National Report on Animal Genetic Resources Indonesia: A strategic Policy Document. The molecular phylogenetic signature of Bali cattle revealed by maternal and paternal markers. 2 Nijman IJ. Romaino SM.. Acknowledgements The authors gratefully acknowledge the research development grant by the Research and Community Service Bureau Universitas Gadjah Mada to support this publication through contract no. 6 Syed-Shabthar SM. Heredity 2003. Maniatis T. Susilawati T. 7 Sambrook J. Heredity 2003. in Madura cross (Limousin x Madura). Lenstra JA. Paternally inherited markers in bovine hybrid populations. Rollinson DHL. Lenstra JA. Restriction fragment of Madura cattle population using PstI and BfaI restriction enzyme Restriction Enzymes Sample Type Location Number PstI BfaI 2 Band 3 Band 1 Band Madura Cattle Pamekasan 17 17 . Hybridization of Banteng (bos Javanicus) and Zebu (Bos Indicus) Revealed by Mitocondrial DNA. Cloning and characterization of bovine SRY gene. 93: 89–100. Molecular Cloning. Mahani MC. 10 Payne WJA. Shamshad E. Mohd-Zin NA. The origin of Indonesian cattle and conservation genetics of the Bali cattle breed. de Ruijter C. A Laboratory Manual. Van Tol HTA. 2003. Cui X. 1989.Zainuddin R. all the origin of Madura cattle (domesticated Madura cattle and conserved Madura cattle) indicated having only one fragment. Fazly-Ann ZA. Z Tierzuch Zu chtsbiol 1976. Previously studies indicated BfaI PCR–RFLP of SRY from the bovine species. Ochieng JY.81%) Total Sample 43 43 15 28 As a result. Andersson G. Bali cattle and Madura cattle that the nucleotide sequences as well as the corresponding BfaI cleavage patterns of banteng and Bali cattle were identical and different from other bovine species [2]. However. 23(3): 145-148. Verkaar ELC. 1986. Itagaki Y. LPPM-UGM/1203/LIT/2013. Gramedia. Madura cattle has the typical zebu hump. Rodriquez-Martinez H. Madura cattle. Satelite DNA. By using BfaI restriction enzyme. Roden C. AFLP and Microsatellite. 66: 994–1000. 5 Mohamad K. Rege JEO. Purwantara B. 4. Anim Sci Technol 1995. 91: 565–569. Romero Mendoza L. Md-Zain BM. 40(8): 5165-76. Hanekamp E. A cytogenetic Investigation of Madura Cattle. Vervaecke H. Sulawati T. Sato S. 17 (100%) (100%) Conserved Madura Cattle Sapudi Island 4 4 . Mol.18%) (31.Rep 2013. / Procedia Environmental Sciences 20 (2014) 365 – 369 369 Table 1. while the mitochondrial DNA was either of zebu or banteng origin [2].. References 1 Pane I. . so the protection and conservation of Madura cattle is therefore urgent. 90(1):10-16. Bavergen MW. Reproduction in Domestic Animals 2007. Reprod Domest Anim 2012. Otsen M. The hybrid origin of the Madura cattle nuclear genome has been verified by SFLP and AFLP analyses. Fritsch EF. Abas-Mazni O. Crossbreeding is considered as a threat.. Nijman IJ. 9 Verkaar ELC.