doi:10.1111/j.1365-2052.2007.01602.x Mitochondrial DNA analyses of Indian water buffalo support a distinct genetic origin of river and swamp buffalo S. Kumar, M. Nagarajan, J. S. Sandhu, N. Kumar, V. Behl and G. Nishanth Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India Summary Water buffalo (Bubalus bubalis) is broadly classified into river and swamp categories, but it remains disputed whether these two types were independently domesticated, or if they are the result of a single domestication event. In this study, we sequenced the mitochondrial D- loop region and cytochrome b gene of 217 and 80 buffalo respectively from eight breeds/ locations in northern, north-western, central and southern India and compared our results with published Mediterranean and swamp buffalo sequences. Using these data, river and swamp buffalo were distinguished into two distinct clades. Based upon the existing knowledge of cytogenetic, ecological and phenotypic parameters, molecular data and present-day distribution of the river and swamp buffalo, we suggest that these two types were domesticated independently, and that classification of the river and swamp buffalo as two related subspecies is more appropriate. Keywords divergence, domestication, mitochondrial DNA, river buffalo, swamp buffalo. Introduction The domestic water buffalo is a species of paramount importance to many Asian countries, particularly to those in the Indian subcontinent. This species is a major source of milk, meat, draft power, hide and employment to marginal farmers and landless labourers in many Asian countries. In spite of the tremendous economic importance of buffalo, interest in buffalo genomics has been limited (Moore et al. 1995; Barker et al. 1997; Navani et al. 2002; Iannuzzi et al. 2003; Kierstein et al. 2004; Kumar et al. 2006). The domestic water buffalo are broadly classified into two major categories based upon their phenotype, behaviour and karyotype: river buffalo (2n ¼ 50) found in the Indian subcontinent, Middle East and Eastern Europe and swamp buffalo (2n ¼ 48) distributed in China, Bangladesh, the Southeast Asian countries and north-eastern states of India (Cockrill 1981). The time and place of domestication of this species has not yet been resolved. It is believed that buffalo was domesticated in the Indus civilization, the Yangtze and the Euphrates and Tigris some 5000 years ago (Cockrill 1981). However, Chen & Li (1989) proposed that the domestication of this species occurred in China during the fifth millennium BC. Two contradictory suggestions have been proposed: that these two types were domesticated independently (Tanaka et al. 1996; Lau et al. 1998) and that these are a product of a single domestication event (Kierstein et al. 2004). Kierstein et al. (2004) analysed the mitochondrial (mt) D-loop DNA sequences of Indian river and swamp (Carabao) buffalo from Brazil and Mediterra- nean animals from Italy. They suggested that the river and swamp buffalo were the product of a single domestication event probably in the Indian subcontinent. Earlier, Kikkawa et al. (1997) suggested that the domestic river and swamp lines arose independently based on differences in their cytochrome b sequences. Although India is a hotspot of buffalo biodiversity (George et al. 1988), and it is generally believed that buffalo were domesticated in India (Cockrill 1981), none of the studies dealing with buffalo domestica- tion (Kikkawa et al. 1997; Lau et al. 1998; Kierstein et al. 2004) sampled animals from Indian locations. Here we report the sequence analysis of the mt D-loop region (n ¼ 217) and the cytochrome b gene (n ¼ 80) in Indian river buffalo and compare them with published sequences of 10 Address for correspondence Dr Satish Kumar, National Facility for Transgenic and Gene Knockout Mice, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. E-mail: satishk@ccmb.res.in Accepted for publication 29 January 2007 Ó 2007 The Authors, Journal compilation Ó 2007 International Society for Animal Genetics, Animal Genetics, 38, 227–232 227