Polymorphism and evolutionary profile of mitochondrial DNA control region inferred from the sequences of Pakistani goats

INTRODUCTION

The control region (displacement loop: D-loop) is the most variable and non-coding portion of the mammalian mitochondrial genome (Cann et al. 1984; Wilson et al. 1985). This region controls the mitochondrial DNA (mtDNA) replication by regulating the activities of various enzymes and proteins that are coded by the nuclear genes (Ghivizzani et al. 1993; Nass 1995 It contains the origin of heavy strand and the promoters for the light and heavy strand replication (Anderson et al. 1982; Chang et al. 1985). In Asia, Pakistan is one of the largest countries in goat production, approximately 36 million head (Hasnain 1985). However, there are only a few studies on the molecular genetic variability and phylogenetic work in domestic goats using the complete (Takada et al. 1997; Mannen et al. 2001; Sultana et al. 2003) or partial mtDNA control region sequences (Luikart et al. 2001). Particularly, the evolutionary pattern of the caprine control region has not been studied yet. In our previous study, we have observed extensive mtDNA diversity in Pakistani domestic goats by finding four distinct mtlineages A, B, C and D (Sultana et al. 2003). In the present study, we investigated the mtDNA control region polymorphisms in Pakistani goats to gain deeper insight into their diversity and compared the organization of the caprine mtDNA control region with those of other mammalian species. More than 10,000 years ago, the transition of humans from
hunting to the manipulation of the behavior of certain animals lead to the process of domestication ( Brock J- Clutton 1999). This process contributed to the rise of human civilization by enabling people to settle into a sedentary lifestyle. The goat was one of the first domesticated animals ( V , Porter.1996)(M, Zeder et al. 2006). It was a source of milk, meat, dung for fuel and materials for clothing and building such as hair, bone and skin ( Brock J-

Clutton 1999) .( D, MacHugh, D, Bradley.2001) oat breeding is an important resource in developing countries where small ruminants are a source of human nutrition and an integral part of rural farming. This species has good productive performances and an excellent ability to adapt to a wide range of climatic conditions (McDowell & Woodward

1982; Devendra 1999). To date, sequences from many species are known and the complete sequence of goat mitochondrial genome (Accession number: GenBank AF533441) was deposited in 2003 (Parma et al. 2003). Many studies used mtDNA as an important means of population studies.( G,Luikart et al . 2001) made the first important research.( Naderi et al.2007) using a large mtDNA analysis, identified six haplogroups mtDNA. The origin was confirmed by genetic studies based on mitochondrial(T, Takada et al. 1997) ( V, Manceau et al.1999) and nuclear DNA( N, Pidancier et al.2006).Mitochondrial DNA is commonly used for the study of domesticated species. The control region has been especially used for describing the genetic polymorphism of goats (G, Luikart et al . 2001) because it is variable and structured enough across the geographical range of the species, and evolves at a constant rate(M, Bruford et al 2003) Moreover, it allows maternal lineages to be followed and is less sensitive to introgression from wild species than nuclear DNA (G, Luikart et al . 2001). However, studies on nuclear genes are needed because they give information on gene flow and selection processes that had a great influence on the evolution of livestock species (M, Bruford et al 2003)
Question How much D LOOP mtDNA differ in expression from one goat species to other goat species? To identify how much haplogroups are present for further study? How mtDNA controlling region interfere on evolutionary profile in Pakistani goats?

MATERIALS AND METHODS Sample collection Hair root samples of 232 goats were collected from a slaughterhouse in Pakistan; 152 from 13 pure Pakistani caprine breeds and 80 random samples. Amplification, purification and DNA sequencing Complete mtDNA control regions (1213 bp) of 30 domestic goats were directly amplified and sequenced from hair roots as described by Sultana et al. (2003).The 17 bp deletion and 76 bp

insertion events were examined in a total of 232 domestic goats by PCR amplification. The primer set (F; 5-CAACACAAACT TCCCA CTCCACAA-3 and R; 5-GTATTTGTGTGTAG GCGAGCGGTG-3) was applied to amplify a 113 bp fragment that includes the 17 bp deletion. The PCR for 17 bp deletion was performed in 10 L reactions containing 5 ng of the genomic DNA purified from hair roots, 2.5 mmol/L MgCl2, 200 mol/L of each dNTP, 1 mol/L of each primer and 1 unit of EX Taq polymerase (Takara Shuzo, Tokyo, Japan). After 2 min preheating at 95C, the PCR reactions underwent 30 cycles of 30 s denaturation at 95C, 30 s annealing at 58C and 1 min extension at 72C followed by extension for 7 min at 72C. The primer set (F; 5-CAACAC AAACTTCCCACTCCACAA-3 and R: 5CTACAATTT ATGCTCCG GGTCG-3) was used to amplify a 583 bp segment containing 76 bp insertion. The PCR conditions for 76 bp insertion were prior heating for 2 min at 95C, which was then followed by 30 cycles of 30 s denaturation at 95C, 30 s annealing at 55C and 1 min extension at 72C with a final extension for 7 min on completion of the last cycle. The PCR products were detected on 2.5% agarose gels Sequence analysis and polymorphism detections Alignment of the mtDNA control region sequences of 30 Pakistani goats with the reference sequence (DDBJ/GenBank/EMBL accession number: AB044295) was determined using CLUSTAL W (Thompson et al. 1994). The structural and functional features of the caprine control region were deduced from the established representative sequences of cattle (Anderson et al. 1982), humans (Anderson et al. 1981), sheep (Hiendleder et al. 1998b) and pigs (Okumura et al. 1996). The published sequences with least numbers of repeat motifs and insertions/deletions were chosen from the GenBank for comparison. The terminology criteria for the control region applied are according to Saccone et al. (1991). REFERENCES 1. S, Anderson, AT, Bankler, BG, Barrell, Bruijn MHL De, AR, Coulson, J, Drouin, IC Eperon,, DP, Nierlich, BA, Roe , F, Sanger , PH, Schreier, AJH Smith, R, , Staden , IG, Young. 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457465. 2. S, Anderson, MHL, De ,Bruijin , AR, Colson ,IC, Eperon , F, Sanger IG ,Young. 1982. Complete sequence of bovine mitochondrial DNA conserved features of the mammalian mitochondrial genome. Journal of Molecular Biology 156, 683692.

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