The 4th International Conference

Sustainable Future for Human Security Kyoto, 18-21 October 2013

Analysis of DNA Polymorphism in SRY Gene of Madura Cattle Populations

T. Hartatika*. T.S.M. Widib, S.D. Volkandaria, Kanazawa Takuyac and Sumadia aLaboratory of Animal Breeding, Faculty of Animal Science, UGM, Yogyakarta, 55281, Indonesia bLaboratory of Draught and Companion Animal, Faculty of Animal Science, UGM, Yogyakarta, 55281, Indonesia cLaboratory of Animal Cell and Engineering, College of Agriculture, Ibaraki University, Japan

Abstract

The aim of this research is to assess the SRY-gene polymorphism in Madura cattle populations and the hybrids of Madura cow with Limousine bull. Here, we investigate the specific markers of the paternally transmitted Y-chromosome for the direct detection of male-mediated introgression.We used PCR–RFLP to differentiate the Y-chromosomes of Madura cattle, and Limousin x Madura crossbred cattle. The result shows that the introduction of Limousin bull changes the Y-chromosome proportion 73.68%. The fantastic number of changing should become the serious attention to preserve of pure Madura cattle as national germ plasm in Indonesia.

© 2013 SustaiN Society

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

Abbreviations:

DNA Deoxyribonucleic acid PCR Polymerase Chain Reaction RFLP Restriction Fragment Length Polymorphism

1. Introduction Madura cattle is one of indigenous breed maintained in Madura region of Indonesia. Madura cattle is the same as Javanese cattle which has previously lived in Java Island then migrate to Madura Island [1, 2]. Therefore, in the course of adaptation to the local climate and geographical condition, finally

* Corresponding author. Tel.: +62-274-4333373; fax: +62-274-521578.

E-mail address: tetyharuta@yahoo.com.

Tety Hartatik/ the 4th SustaiN 2013 Conference

geographical isolates of Javanese cattle in Madura Island became Madura cattle as recently been reported. The previous study [2] have analyzed DNA from zebu, dan, banteng and Madura cattle, and have found Madura cattle to have characteristics of Zebu and Banteng. The uniformity of this breed was created through a continuous selection by the people at Madura [3]. Since 2001, crossbreeding with Limousin is allowed. Then, Limousin is preferred by Maduranesse due to its red colour as Madura cattle. In restricted area of Madura, Sumenep Regency, 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 [personal communication]. Artificial crossbreeding and naturally-occurring introgression leads to both the loss of indigenous breeds and the loss of adaptation potentials to local environment. It may have an impact on the formation of local breeds, and could also affect the genetic integrity of them. Y-Chromosome is useful for analyzing the paternal lineages of the crossbred cattle population (e.g. Limousin x Madura). Therefore, we investigate the DNA polymorphism of SRY genes on Y-chromosome of Madura hybrid cattle, using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The aim of this research is to assess the SRY-gene polymorphism in Madura cattle populations and the hybrids of Madura cow with Limousine bull and to protect uncontrolled crossbreeding in specific area of pure Madura cattle.

2. Material and Methods

2.1. Sample collection and DNA extraction

Three type cattle consisted of Madura cattle, and Limousin x Madura (crossbred cattle, were used in this research. DNA was isolated from their blood samples by using standard SDS/proteinase K extraction [4]. Isolation and analysis DNA samples were conducted at Laboratory of Animal Breeding, Faculty of Animal Science, Gadjah Mada University.

2.2. PCR-RFLP

The numbering of primer binding sites and restriction sites referred to the Genbank entry AB039748 for bovine SRY [5]. For detection of the Madura hybrid cattle Y-chromosome, we used 50 ng genomic DNA in a total volume of 25 ml and perform the PCR using one of two pairs of primers: SRY-F2 (50-GTT GAT GGG TTT GGG CTG ACT) and SRY-R2 (50-AAA TTG AGA TAA AGA GCG CCT) with 1.5 mM MgCl2, 0.2 mM dNTP and 1.25U Taq DNA polymerase (Promega, Madison, WI, USA). The program consisted of an initial denaturation for 2 min at 95°C followed by 30 cycles of 15 s at 92°C, 30 s at 60°C and 45 s at 72°C, and by a final extension for 5 min at 72°C. PCR products were first separated by electrophoresis on a 1% agarose gel, then 5 ml of the PCR product was digested by the addition of 5–10U of either PstI or BfaI restriction enzyme (New England Biolabs, Beverley, MA, USA) and the reaction buffer of manufacture’s recommendation. The samples were digested for 3 h at 37°C and were electrophoresed on a 12% polyacrylamide gel to analyze restriction-fragments.

Tety Hartatik/ the 4th SustaiN 2013 Conference

3. Results and Discussion The result of DNA amplification by PCR method produced 928 bp as illustrated on Figure 1. 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. The result of this study shows that both of Madura cattle in Pamekasan district and Sapudi island has one pattern of restriction site. While the different result was showed on hybrid of Limousin bull and Madura dam. Where the PstI pattern has one type of restriction, while BfaI pattern has two types of restriction (Table 1).

Figure 1. A 928 bp sequence of SRY gene of bovine (NCBI: AB039748)

And location of restriction site of PstI enzyme (5’-C˄TGCA˅

G-3’) and BfaI (5’-C˅

TA˄G-3’)

The occurrence of different type of Y Chromosome on Limousin x Madura crossbred cattle showed the contribution of Bos taurus gene through the paternal line. There are 73,68% change of sequence type of Y Chromosome in crossbred cattle. The number showed that the consistent using of Limousin bull continually can shifting of genetic trait from Y Chromosome on local cattle.

Previouse study described the sequence variation in the Y-chromosomal SRY gene for the design of convenient

tests of paternal lineages in bovine populations. SRY is the most obvious target for these assays, since it is the

only single-copy gene without an X-chromosomal homologue [6]. BfaI PCR–RFLP of SRY from the bovine species, Bali cattle and Madura cattle was checked by Veekar et al which showed 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. Madura cattle has the typical zebu hump, but is also supposed to

result from zebu–banteng hybridization [7]. The hybrid origin of the Madura nuclear genome has been verified by SFLP and AFLP analyses, while the mitochondrial DNA was either of zebu or banteng origin [2].

Tety Hartatik/ the 4th SustaiN 2013 Conference

Table 1. Restriction fragment of Madura cattle population using PstI and BfaI restriction Enzyme

Sample Type Location Number Restriction Enzymes

Pst I BfaI

2 Band 3 Band 1 Band

Madura Cattle Pamekasan 10 10 (100%)

- 10 (100%)

Pure Madura Cattle Sapudi Island 4 4 (100%)

- 4 (100%)

Limousin x Madura Cattle Pamekasan 19 19 (100%)

14 (73.68%)

5 (26.32%)

Total Sample 33 33 14 19

4. Conclussion

The introduction of Limousin bulls semen has changed the proportion of Y Chromosome that reach the differences of RE type 73,68% than the previouse type. The fantastic number of changing should become the serious attention to preserve of pure Madura cattle as national germ plasm in Indonesia.

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